4(phenyl-piperazinyl-methyl) benzamide derivatives and their use for the treatment of pain or gastrointestinal disorders

ABSTRACT

Compounds of general formula: 
                         
wherein R 1 , R 2  and R 3  are as defined in the specification, as well as salts, enantiomers thereof and pharmaceutical compositions including the compounds are prepared. They are useful in therapy, in particular in the management of pain.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/533,654, now U.S. Pat. No. 7,253,173, filed May 4, 2005, which wasthe National Stage of International Application No. PCT/SE03/01707,which was filed Nov. 5, 2003, which claims priority under 35 U.S.C 365and 35 U.S.C. §119 (a)-(d) to Swedish application no. SE 0203303-3,filed Nov. 7, 2002.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is directed to novel compounds, to a process fortheir preparation, their use and pharmaceutical compositions comprisingthe novel compounds. The novel compounds are useful in therapy, and inparticular for the treatment of pain, anxiety and functionalgastrointestinal disorders.

2. Discussion of Relevant Art

The receptor has been identified as having a role in many bodilyfunctions such as circulatory and pain systems. Ligands for the δreceptor may therefore find potential use as analgesics, and/or asantihypertensive agents. Ligands for the δ receptor have also been shownto possess immunomodulatory activities.

The identification of at least three different populations of opioidreceptors (μ, δ and κ) is now well established and all three areapparent in both central and peripheral nervous systems of many speciesincluding man. Analgesia has been observed in various animal models whenone or more of these receptors has been activated.

With few exceptions, currently available selective opioid δ ligands arepeptidic in nature and are unsuitable for administration by systemicroutes. One example of a non-peptidic δ-agonist is SNC80 (Bilsky E. J.et al., Journal of Pharmacology and Experimental Therapeutics, 273(1),pp. 359-366 (1995)).

Many δ agonist compounds that have been identified in the prior art havemany disadvantages in that they suffer from poor pharmacokinetics andare not analgesic when administered by systemic routes. Also, it hasbeen documented that many of these δ agonist compounds show significantconvulsive effects when administered systemically.

U.S. Pat. No. 6,130,222 to Roberts et al. describes some δ-agonists.

However, there is still a need for improved δ-agonists.

DESCRIPTION OF THE INVENTION

Definitions

Unless specified otherwise within this specification, the nomenclatureused in this specification generally follows the examples and rulesstated in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F,and H, Pergamon Press, Oxford, 1979, which is incorporated by referencesherein for its exemplary chemical structure names and rules on namingchemical structures.

The term “C_(m-n)” or “C_(m-n) group” used alone or as a prefix, refersto any group having m to n carbon atoms.

The term “hydrocarbon” used alone or as a suffix or prefix, refers toany structure comprising only carbon and hydrogen atoms up to 14 carbonatoms.

The term “hydrocarbon radical” or “hydrocarbyl” used alone or as asuffix or prefix, refers to any structure as a result of removing one ormore hydrogens from a hydrocarbon.

The term “alkyl” used alone or as a suffix or prefix, refers tomonovalent straight or branched chain hydrocarbon radicals comprising 1to about 12 carbon atoms.

The term “alkylene” used alone or as suffix or prefix, refers todivalent straight or branched chain hydrocarbon radicals comprising 1 toabout 12 carbon atoms, which serves to links two structures together.

The term “alkenyl” used alone or as suffix or prefix, refers to amonovalent straight or branched chain hydrocarbon radical having atleast one carbon-carbon double bond and comprising at least 2 up toabout 12 carbon atoms.

The term “alkynyl” used alone or as suffix or prefix, refers to amonovalent straight or branched chain hydrocarbon radical having atleast one carbon-carbon triple bond and comprising at least 2 up toabout 12 carbon atoms.

The term “cycloalkyl,” used alone or as suffix or prefix, refers to amonovalent ring-containing hydrocarbon radical comprising at least 3 upto about 12 carbon atoms.

The term “cycloalkenyl” used alone or as suffix or prefix, refers to amonovalent ring-containing hydrocarbon radical having at least onecarbon-carbon double bond and comprising at least 3 up to about 12carbon atoms.

The term “cycloalkynyl” used alone or as suffix or prefix, refers to amonovalent ring-containing hydrocarbon radical having at least onecarbon-carbon triple bond and comprising about 7 up to about 12 carbonatoms.

The term “aryl” used alone or as suffix or prefix, refers to amonovalent hydrocarbon radical having one or more polyunsaturated carbonrings having aromatic character, (e.g. 4 n+2 delocalized electrons) andcomprising 5 up to about 14 carbon atoms.

The term “arylene” used alone or as suffix or prefix, refers to adivalent hydrocarbon radical having one or more polyunsaturated carbonrings having aromatic character, (e.g. 4 n+2 delocalized electrons) andcomprising 5 up to about 14 carbon atoms, which serves to link twostructures together.

The term “heterocycle” used alone or as a suffix or prefix, refers to aring-containing structure or molecule having one or more multivalentheteroatoms, independently selected from N, O, P and S, as a part of thering structure and including at least 3 and up to about 20 atoms in thering(s). Heterocycle may be saturated or unsaturated, containing one ormore double bonds, and heterocycle may contain more than one ring. Whena heterocycle contains more than one ring, the rings may be fused orunfused. Fused rings generally refer to at least two rings share twoatoms therebetween. Heterocycle may have aromatic character or may nothave aromatic character.

The term “heteroaromatic” used alone or as a suffix or prefix, refers toa ring-containing structure or molecule having one or more multivalentheteroatoms, independently selected from N, O, P and S, as a part of thering structure and including at least 3 and up to about 20 atoms in thering(s), wherein the ring-containing structure or molecule has anaromatic character (e.g., 4 n+2 delocalized electrons).

The term “heterocyclic group,” “heterocyclic moiety,” “heterocyclic,” or“heterocyclo” used alone or as a suffix or prefix, refers to a radicalderived from a heterocycle by removing one or more hydrogens therefrom.

The term “heterocyclyl” used alone or as a suffix or prefix, refers amonovalent radical derived from a heterocycle by removing one hydrogentherefrom.

The term “heterocyclylene” used alone or as a suffix or prefix, refersto a divalent radical derived from a heterocycle by removing twohydrogens therefrom, which serves to links two structures together.

The term “heteroaryl” used alone or as a suffix or prefix, refers to aheterocyclyl having aromatic character.

The term “heterocylcoalkyl” used alone or as a suffix or prefix, refersto a heterocyclyl that does not have aromatic character.

The term “heteroarylene” used alone or as a suffix or prefix, refers toa heterocyclylene having aromatic character.

The term “heterocycloalkylene” used alone or as a suffix or prefix,refers to a heterocyclylene that does not have aromatic character.

The term “six-membered” used as prefix refers to a group having a ringthat contains six ring atoms.

The term “five-membered” used as prefix refers to a group having a ringthat contains five ring atoms.

A five-membered ring heteroaryl is a heteroaryl with a ring having fivering atoms wherein 1, 2 or 3 ring atoms are independently selected fromN, O and S.

Exemplary five-membered ring heteroaryls are thienyl, furyl, pyrrolyl,imidazolyl, thiazolyl, oxazolyl, pyrazolyl, isothiazolyl, isoxazolyl,1,2,3-triazolyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl,1,2,4-triazolyl, 1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4-oxadiazolyl.

A six-membered ring heteroaryl is a heteroaryl with a ring having sixring atoms wherein 1, 2 or 3 ring atoms are independently selected fromN, O and S.

Exemplary six-membered ring heteroaryls are pyridyl, pyrazinyl,pyrimidinyl, triazinyl and pyridazinyl.

The term “substituted” used as a prefix refers to a structure, moleculeor group, wherein one or more hydrogens are replaced with one or moreC₁₋₆hydrocarbon groups, or one or more chemical groups containing one ormore heteroatoms selected from N, O, S, F, Cl, Br, I, and P. Exemplarychemical groups containing one or more heteroatoms include —NO₂, —OR,—Cl, —Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH, —NHR, —NR₂, —SR,—SO₃H, —SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂, —NRC(═O)R, oxo(═O), imino (═NR), thio (═S), and oximino (═N—OR), wherein each “R” is aC₁₋₆hydrocarbyl. For example, substituted phenyl may refer tonitrophenyl, methoxyphenyl, chlorophenyl, aminophenyl, etc., wherein thenitro, methoxy, chloro, and amino groups may replace any suitablehydrogen on the phenyl ring.

The term “substituted” used as a suffix of a first structure, moleculeor group, followed by one or more names of chemical groups refers to asecond structure, molecule or group, which is a result of replacing oneor more hydrogens of the first structure, molecule or group with the oneor more named chemical groups. For example, a “phenyl substituted bynitro” refers to nitrophenyl.

Heterocycle includes, for example, monocyclic heterocycles such as:aziridine, oxirane, thiirane, azetidine, oxetane, thietane, pyrrolidine,pyrroline, imidazolidine, pyrazolidine, pyrazoline, dioxolane, sulfolane2,3-dihydrofuran, 2,5-dihydrofuran tetrahydrofuran, thiophane,piperidine, 1,2,3,6-tetrahydro-pyridine, piperazine, morpholine,thiomorpholine, pyran, thiopyran, 2,3-dihydropyran, tetrahydropyran,1,4-dihydropyridine, 1,4-dioxane, 1,3-dioxane, dioxane, homopiperidine,2,3,4,7-tetrahydro-1H-azepine homopiperazine, 1,3-dioxepane,4,7-dihydro-1,3-dioxepin, and hexamethylene oxide.

In addition, heterocycle includes aromatic heterocycles, for example,pyridine, pyrazine, pyrimidine, pyridazine, thiophene, furan, furazan,pyrrole, imidazole, thiazole, oxazole, pyrazole, isothiazole, isoxazole,1,2,3-triazole, tetrazole, 1,2,3-thiadiazole, 1,2,3-oxadiazole,1,2,4-triazole, 1,2,4-thiadiazole, 1,2,4-oxadiazole, 1,3,4-triazole,1,3,4-thiadiazole, and 1,3,4-oxadiazole.

Additionally, heterocycle encompass polycyclic heterocycles, forexample, indole, indoline, isoindoline, quinoline, tetrahydroquinoline,isoquinoline, tetrahydroisoquinoline, 1,4-benzodioxan, coumarin,dihydrocoumarin, benzofuran, 2,3-dihydrobenzofuran, isobenzofuran,chromene, chroman, isochroman, xanthene, phenoxathiin, thianthrene,indolizine, isoindole, indazole, purine, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, phenanthridine,perimidine, phenanthroline, phenazine, phenothiazine, phenoxazine,1,2-benzisoxazole, benzothiophene, benzoxazole, benzthiazole,benzimidazole, benztriazole, thioxanthine, carbazole, carboline,acridine, pyrolizidine, and quinolizidine.

In addition to the polycyclic heterocycles described above, heterocycleincludes polycyclic heterocycles wherein the ring fusion between two ormore rings includes more than one bond common to both rings and morethan two atoms common to both rings. Examples of such bridgedheterocycles include quinuclidine, diazabicyclo[2.2.1]heptane and7-oxabicyclo[2.2.1]heptane.

Heterocyclyl includes, for example, monocyclic heterocyclyls, such as:aziridinyl, oxiranyl, thiiranyl, azetidinyl, oxetanyl, thietanyl,pyrrolidinyl, pyrrolinyl, imidazolidinyl, pyrazolidinyl, pyrazolinyl,dioxolanyl, sulfolanyl, 2,3-dihydrofuranyl, 2,5-dihydrofuranyl,tetrahydrofuranyl, thiophanyl, piperidinyl,1,2,3,6-tetrahydro-pyridinyl, piperazinyl, morpholinyl, thiomorpholinyl,pyranyl, thiopyranyl, 2,3-dihydropyranyl, tetrahydropyranyl,1,4-dihydropyridinyl, 1,4-dioxanyl, 1,3-dioxanyl, dioxanyl,homopiperidinyl, 2,3,4,7-tetrahydro-1H-azepinyl, homopiperazinyl,1,3-dioxepanyl, 4,7-dihydro-1,3-dioxepinyl, and hexamethylene oxidyl.

In addition, heterocyclyl includes aromatic heterocyclyls or heteroaryl,for example, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, thienyl,furyl, furazanyl, pyrrolyl, imidazolyl, thiazolyl, oxazolyl, pyrazolyl,isothiazolyl, isoxazolyl, 1,2,3-triazolyl, tetrazolyl,1,2,3-thiadiazolyl, 1,2,3-oxadiazolyl, 1,2,4-triazolyl,1,2,4-thiadiazolyl, 1,2,4-oxadiazolyl, 1,3,4-triazolyl,1,3,4-thiadiazolyl, and 1,3,4oxadiazolyl.

Additionally, heterocyclyl encompasses polycyclic heterocyclyls(including both aromatic or non-aromatic), for example, indolyl,indolinyl, isoindolinyl, quinolinyl, tetrahydroquinolinyl,isoquinolinyl, tetrahydroisoquinolinyl, 1,4-benzodioxanyl, coumarinyl,dihydrocoumarinyl, benzofuranyl, 2,3-dihydrobenzofuranyl,isobenzofuranyl, chromenyl, chromanyl, isochromanyl, xanthenyl,phenoxathiinyl, thianthrenyl, indolizinyl, isoindolyl, indazolyl,purinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl,cinnolinyl, pteridinyl, phenanthridinyl, perimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, phenoxazinyl, 1,2-benzisoxazolyl,benzothiophenyl, benzoxazolyl, benzthiazolyl, benzimidazolyl,benztriazolyl, thioxanthinyl, carbazolyl, carbolinyl, acridinyl,pyrolizidinyl, and quinolizidinyl.

In addition to the polycyclic heterocyclyls described above,heterocyclyl includes polycyclic heterocyclyls wherein the ring fusionbetween two or more rings includes more than one bond common to bothrings and more than two atoms common to both rings. Examples of suchbridged heterocycles include quinuclidinyl, diazabicyclo[2.2.1]heptyl;and 7-oxabicyclo[2.2.1]heptyl.

The term “alkoxy” used alone or as a suffix or prefix, refers toradicals of the general formula —O—R, wherein R is selected from ahydrocarbon radical. Exemplary alkoxy includes methoxy, ethoxy, propoxy,isopropoxy, butoxy, t-butoxy, isobutoxy, cyclopropylmethoxy, allyloxy,and propargyloxy.

The term “amine” or “amino” used alone or as a suffix or prefix, refersto radicals of the general formula —NRR′, wherein R and R′ areindependently selected from hydrogen or a hydrocarbon radical.

Halogen includes fluorine, chlorine, bromine and iodine.

“Halogenated,” used as a prefix of a group, means one or more hydrogenson the group is replaced with one or more halogens.

“RT” or “rt” means room temperature.

DESCRIPTION OF EMBODIMENTS

In one aspect, the invention provides a compound of formula I, apharmaceutically acceptable salt thereof, diastereomers thereof,enantiomers thereof, and mixtures thereof:

wherein

R¹ is selected from —H, C₆₋₁₀aryl, C₂₋₆heteroaryl, C₆₋₁₀aryl-C₁₋₄alkyl,and C₂₋₆heteroaryl-C₁₋₄alkyl, wherein said C₆₋₁₀aryl, C₂₋₆heteroaryl,C₆₋₁₀aryl-C₁₋₄alkyl, and C₂₋₆heteroaryl-C₁₋₄alkyl are optionallysubstituted with one or more groups selected from —R, —NO₂, —OR, —Cl,—Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H,—SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂, —NRC(═O)R, and—NRC(═O)—OR, wherein R is, independently, a hydrogen or C₁₋₆alkyl;

R² is selected from —H, C₁₋₆alkyl and C₃₋₆cycloalkyl, wherein saidC₁₋₆alkyl and C₃₋₆cycloalkyl are optionally substituted with one or moregroups selected from —OR, —Cl, —Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH,—NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H, —SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR,—C(═O)NR₂, —NRC(═O)R, and —NRC(═O)—OR, wherein R is, independently, ahydrogen or C₁₋₆alkyl; and

R³ is selected from C₁₋₆alkyl and C₃₋₆cycloalkyl, wherein said C₁₋₆alkyland C₃₋₆cycloalkyl are optionally substituted with one or more groupsselected from —OR, —Cl, —Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH,—NHR, —NR₂, —SR, —SO₃H, —SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂,—NRC(═O)R, and —NRC(═O)—OR, wherein R is, independently, a hydrogen orC₁₋₆alkyl.

In another embodiment, the compounds of the present invention are thoseof formula I, wherein

R¹ is —CH₂—R⁴, wherein R⁴ is selected from phenyl; pyridyl; thienyl;furyl; imidazolyl; triazolyl; pyrrolyl; thiazolyl; and N-oxido-pyridyl,wherein said phenyl; pyridyl; thienyl; furyl; imidazolyl; triazolyl;pyrrolyl; thiazolyl; and N-oxido-pyridyl are optionally substituted withone or more groups selected from C₁₋₆alkyl, halogenated C₁₋₆alkyl, —NO₂,—CF₃, C₁₋₆ alkoxy, chloro, fluoro, bromo, and iodo;

R² is selected from —H and C₁₋₃alkyl; and

R³ is selected from C₁₋₆alkyl, and C₃₋₆cycloalkyl.

In another embodiment, the compounds of the present invention are thoseof formula I, wherein R¹ is —CH₂—R⁴, wherein R⁴ is selected from phenyl;pyridyl; thienyl; furyl; imidazolyl; pyrrolyl and thiazolyl;

R² is selected from —H and methyl; and

R³ is selected from methyl, ethyl, propyl and isopropyl.

In a further embodiment, the compounds of the present invention arethose of formula I, wherein R¹ is —H;

R² is selected from —H and C₁₋₃alkyl; and

R³ is selected from C₁₋₆alkyl, and C₃₋₆cycloalkyl.

It will be understood that when compounds of the present inventioncontain one or more chiral centers, the compounds of the invention mayexist in, and be isolated as, enantiomeric or diastereomeric forms, oras a racemic mixture. The present invention includes any possibleenantiomers, diastereomers, racemates or mixtures thereof, of a compoundof Formula I. The optically active forms of the compound of theinvention may be prepared, for example, by chiral chromatographicseparation of a racemate, by synthesis from optically active startingmaterials or by asymmetric synthesis based on the procedures describedthereafter.

It will also be appreciated that certain compounds of the presentinvention may exist as geometrical isomers, for example E and Z isomersof alkenes. The present invention includes any geometrical isomer of acompound of Formula I. It will further be understood that the presentinvention encompasses tautomers of the compounds of the formula I.

It will also be understood that certain compounds of the presentinvention may exist in solvated, for example hydrated, as well asunsolvated forms. It will further be understood that the presentinvention encompasses all such solvated forms of the compounds of theformula I.

Within the scope of the invention are also salts of the compounds of theformula I. Generally, pharmaceutically acceptable salts of compounds ofthe present invention may be obtained using standard procedures wellknown in the art, for example by reacting a sufficiently basic compound,for example an alkyl amine with a suitable acid, for example, HCl oracetic acid, to afford a physiologically acceptable anion. It may alsobe possible to make a corresponding alkali metal (such as sodium,potassium, or lithium) or an alkaline earth metal (such as a calcium)salt by treating a compound of the present invention having a suitablyacidic proton, such as a carboxylic acid or a phenol with one equivalentof an alkali metal or alkaline earth metal hydroxide or alkoxide (suchas the ethoxide or methoxide), or a suitably basic organic amine (suchas choline or meglumine) in an aqueous medium, followed by conventionalpurification techniques.

In one embodiment, the compound of formula I above may be converted to apharmaceutically acceptable salt or solvate thereof, particularly, anacid addition salt such as a hydrochloride, hydrobromide, phosphate,acetate, fumarate, maleate, tartrate, citrate, methanesulphonateorp-toluenesulphonate.

The novel compounds of the present invention are useful in therapy,especially for the treatment of various pain conditions such as chronicpain, neuropathic pain, acute pain, cancer pain, pain caused byrheumatoid arthritis, migraine, visceral pain etc. This list shouldhowever not be interpreted as exhaustive.

Compounds of the invention are useful as immunomodulators, especiallyfor autoimmune diseases, such as arthritis, for skin grafts, organtransplants and similar surgical needs, for collagen diseases, variousallergies, for use as anti-tumour agents and anti viral agents.

Compounds of the invention are useful in disease states wheredegeneration or dysfunction of opioid receptors is present or implicatedin that paradigm. This may involve the use of isotopically labelledversions of the compounds of the invention in diagnostic techniques andimaging applications such as positron emission tomography (PET).

Compounds of the invention are useful for the treatment of diarrhea,depression, anxiety and stress-related disorders such as post-traumaticstress disorders, panic disorder, generalized anxiety disorder, socialphobia, and obsessive compulsive disorder, urinary incontinence,premature ejaculation, various mental illnesses, cough, lung oedema,various gastro-intestinal disorders, e.g. constipation, functionalgastrointestinal disorders such as Irritable Bowel Syndrome andFunctional Dyspepsia, Parkinson's disease and other motor disorders,traumatic brain injury, stroke, cardioprotection following myocardialinfarction, spinal injury and drug addiction, including the treatment ofalcohol, nicotine, opioid and other drug abuse and for disorders of thesympathetic nervous system for example hypertension.

Compounds of the invention are useful as an analgesic agent for useduring general anaesthesia and monitored anaesthesia care. Combinationsof agents with different properties are often used to achieve a balanceof effects needed to maintain the anaesthetic state (e.g. amnesia,analgesia, muscle relaxation and sedation). Included in this combinationare inhaled anesthetics, hypnotics, anxiolytics, neuromuscular blockersand opioids.

Also within the scope of the invention is the use of any of thecompounds according to the formula I above, for the manufacture of amedicament for the treatment of any of the conditions discussed above.

A further aspect of the invention is a method for the treatment of asubject suffering from any of the conditions discussed above, whereby aneffective amount of a compound according to the formula I above, isadministered to a patient in need of such treatment.

Thus, the invention provides a compound of formula I, orpharmaceutically acceptable salt or solvate thereof, as hereinbeforedefined for use in therapy.

In a further aspect, the present invention provides the use of acompound of formula I, or a pharmaceutically acceptable salt or solvatethereof, as hereinbefore defined in the manufacture of a medicament foruse in therapy.

In the context of the present specification, the term “therapy” alsoincludes “prophylaxis” unless there are specific indications to thecontrary. The term “therapeutic” and “therapeutically” should becontrued accordingly. The term “therapy” within the context of thepresent invention further encompasses to administer an effective amountof a compound of the present invention, to mitigate either apre-existing disease state, acute or chronic, or a recurring condition.This definition also encompasses prophylactic therapies for preventionof recurring conditions and continued therapy for chronic disorders.

The compounds of the present invention are useful in therapy, especiallyfor the therapy of various pain conditions including, but not limitedto: chronic pain, neuropathic pain, acute pain, back pain, cancer pain,and visceral pain.

In use for therapy in a warm-blooded animal such as a human, thecompound of the invention may be administered in the form of aconventional pharmaceutical composition by any route including orally,intramuscularly, subcutaneously, topically, intranasally,intraperitoneally, intrathoracially, intravenously, epidurally,intrathecally, intracerebroventricularly and by injection into thejoints.

In one embodiment of the invention, the route of administration may beorally, intravenously or intramuscularly.

The dosage will depend on the route of administration, the severity ofthe disease, age and weight of the patient and other factors normallyconsidered by the attending physician, when determining the individualregimen and dosage level at the most appropriate for a particularpatient.

For preparing pharmaceutical compositions from the compounds of thisinvention, inert, pharmaceutically acceptable carriers can be eithersolid and liquid. Solid form preparations include powders, tablets,dispersible granules, capsules, cachets, and suppositories.

A solid carrier can be one or more substances, which may also act asdiluents, flavoring agents, solubilizers, lubricants, suspending agents,binders, or table disintegrating agents; it can also be an encapsulatingmaterial.

In powders, the carrier is a finely divided solid, which is in a mixturewith the finely divided compound of the invention, or the activecomponent. In tablets, the active component is mixed with the carrierhaving the necessary binding properties in suitable proportions andcompacted in the shape and size desired.

For preparing suppository compositions, a low-melting wax such as amixture of fatty acid glycerides and cocoa butter is first melted andthe active ingredient is dispersed therein by, for example, stirring.The molten homogeneous mixture in then poured into convenient sizedmoulds and allowed to cool and solidify.

Suitable carriers are magnesium carbonate, magnesium stearate, talc,lactose, sugar, pectin, dextrin, starch, tragacanth, methyl cellulose,sodium carboxymethyl cellulose, a low-melting wax, cocoa butter, and thelike.

The term composition is also intended to include the formulation of theactive component with encapsulating material as a carrier providing acapsule in which the active component (with or without other carriers)is surrounded by a carrier which is thus in association with it.Similarly, cachets are included.

Tablets, powders, cachets, and capsules can be used as solid dosageforms suitable for oral administration.

Liquid form compositions include solutions, suspensions, and emulsions.For example, sterile water or water propylene glycol solutions of theactive compounds may be liquid preparations suitable for parenteraladministration. Liquid compositions can also be formulated in solutionin aqueous polyethylene glycol solution.

Aqueous solutions for oral administration can be prepared by dissolvingthe active component in water and adding suitable colorants, flavoringagents, stabilizers, and thickening agents as desired. Aqueoussuspensions for oral use can be made by dispersing the finely dividedactive component in water together with a viscous material such asnatural synthetic gums, resins, methyl cellulose, sodium carboxymethylcellulose, and other suspending agents known to the pharmaceuticalformulation art.

Depending on the mode of administration, the pharmaceutical compositionwill preferably include from 0.05% to 99% w (percent by weight), morepreferably from 0.10 to 50% w, of the compound of the invention, allpercentages by weight being based on total composition.

A therapeutically effective amount for the practice of the presentinvention may be determined, by the use of known criteria including theage, weight and response of the individual patient, and interpretedwithin the context of the disease which is being treated or which isbeing prevented, by one of ordinary skills in the art.

Within the scope of the invention is the use of any compound of formulaI as defined above for the manufacture of a medicament.

Also within the scope of the invention is the use of any compound offormula I for the manufacture of a medicament for the therapy of pain.

Additionally provided is the use of any compound according to Formula Ifor the manufacture of a medicament for the therapy of various painconditions including, but not limited to: chronic pain, neuropathicpain, acute pain, back pain, cancer pain, and visceral pain.

A further aspect of the invention is a method for therapy of a subjectsuffering from any of the conditions discussed above, whereby aneffective amount of a compound according to the formula I above, isadministered to a patient in need of such therapy.

Additionally, there is provided a pharmaceutical composition comprisinga compound of Formula I, or a pharmaceutically acceptable salt thereof,in association with a pharmaceutically acceptable carrier.

Particularly, there is provided a pharmaceutical composition comprisinga compound of Formula I, or a pharmaceutically acceptable salt thereof,in association with a pharmaceutically acceptable carrier for therapy,more particularly for therapy of pain.

Further, there is provided a pharmaceutical composition comprising acompound of Formula I, or a pharmaceutically acceptable salt thereof, inassociation with a pharmaceutically acceptable carrier use in any of theconditions discussed above.

In a further aspect, the present invention provides a method ofpreparing a compound of formula I.

In one embodiment, the invention provides a process for preparing acompound of formula II, comprising:

a) reacting a compound of formula III:

with a compound of formula IV

in the presence of benzotriazole; and

b) reacting a product formed in step a) with a compound of formula V toform the compound of formula II,

wherein

R⁸ is selected from C₁₋₆alkyl-O—C(═O)—, C₆₋₁₀aryl-C₁₋₄alkyl, andC₂₋₆heteroaryl-C₁₋₄alkyl, wherein said C₁₋₆alkyl-O—C(═O)—,C₆₋₁₀aryl-C₁₋₄alkyl, and C₂₋₆heteroaryl-C₁₋₄alkyl are optionallysubstituted with one or more groups selected from C₁₋₆alkyl, halogenatedC₁₋₆alkyl, —NO₂, —CF₃, C₁₋₆ alkoxy, chloro, fluoro, bromo, and iodo;

M is selected from Li, Na, K, —ZnX¹, and —MgX¹, wherein X¹ is a halogen;and

R⁹ is selected from hydrogen, —R, —NO₂, —OR, —Cl, —Br, —I, —F, —CF₃,—C(═O)R, —C(═O)OH, —NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H, —SO₂R, —S(═O)R,—CN, —OH, —C(═O)OR, —C(═O)NR₂, —NRC(═O)R, and —NRC(═O)—OR, wherein R is,independently, a hydrogen or C₁₋₆hydrocarbyl.

In another embodiment, the invention provides a process for preparing acompound of formula VII:

comprising:

reacting a compound of formula VIII

with a C₁₋₆alkylcarbamate to form the compound of formula VII,

wherein

R⁸ is selected from C₁₋₆alkyl-O—C(═O)—, C₆₋₁₀aryl-C₁₋₄alkyl, andC₂₋₆heteroaryl-C₁₋₄alkyl, wherein said C₁₋₆alkyl-O—C(═O)—,C₆₋₁₀aryl-C₁₋₄alkyl, and C₂₋₆heteroaryl-C₁₋₄alkyl are optionallysubstituted with one or more groups selected from —OR, —Cl, —Br, —I, —F,—CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H, —SO₂R,—S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂, —NRC(═O)R, and —NRC(═O)—OR,wherein R is, independently, a hydrogen or C₁₋₆alkyl;

X is selected from halogen, triflate, and sulfonamide; and

R¹¹ is a C₁₋₆alkyl.

In a further embodiment, the invention provides a process for preparinga compound of formula X,

comprising:

reacting a compound of formula IX,

with R⁴—CHO to form the compound of formula X,wherein

R⁴ is selected from phenyl; pyridyl; thienyl; furyl; imidazolyl;triazolyl; pyrrolyl; thiazolyl; and N-oxido-pyridyl, wherein saidphenyl; pyridyl; thienyl; furyl; imidazolyl; triazolyl; pyrrolyl;thiazolyl; and N-oxido-pyridyl are optionally substituted with one ormore groups selected from C₁₋₆alkyl, halogenated C₁₋₆alkyl, —NO₂, —CF₃,C₁₋₆ alkoxy, chloro, fluoro, bromo, and iodo;

R² is selected from —H, C₁₋₆alkyl and C₃₋₆cycloalkyl, wherein saidC₁₋₆alkyl and C₃₋₆cycloalkyl are optionally substituted with one or moregroups selected from —OR, —Cl, —Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH,—NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H, —SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR,—C(═O)NR₂, —NRC(═O)R, and —NRC(═O)—OR, wherein R is, independently, ahydrogen or C₁₋₆alkyl; and

R³ is selected from C₁₋₆alkyl and C₃₋₆cycloalkyl, wherein said C₁₋₆alkyland C₃₋₆cycloalkyl are optionally substituted with one or more groupsselected from —OR, —Cl, —Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH,—NHR, —NR₂, —SR, —SO₃H, —SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂,—NRC(═O)R, and —NRC(═O)—OR, wherein R is, independently, a hydrogen orC₁₋₆alkyl.

Particularly, the compounds of the present invention can be preparedaccording to the synthetic routes as exemplified in Schemes 1-7.

Accordingly, a further aspect of the invention is a compound of formulaXI, a pharmaceutically acceptable salt thereof, diastereomers,enantiomers, or mixtures thereof:

wherein

R¹ is selected from —H, C₆₋₁₀aryl, C₂₋₆heteroaryl, C₆₋₁₀aryl-C₁₋₄alkyl,and C₂₋₆heteroaryl-C₁₋₄alkyl, wherein said C₆₋₁₀aryl, C₂₋₆heteroaryl,C₆₋₁₀aryl-C₁₋₄alkyl, and C₂₋₆heteroaryl-C₁₋₄alkyl are optionallysubstituted with one or more groups selected from —R, —NO₂, —OR, —Cl,—Br, —I, —F, —CF₃, —C(═O)R, —C(═O)OH, —NH₂, —SH, —NHR, —NR₂, —SR, —SO₃H,—SO₂R, —S(═O)R, —CN, —OH, —C(═O)OR, —C(═O)NR₂, —NRC(═O)R, and—NRC(═O)—OR, wherein R is, independently, a hydrogen or C₁₋₆alkyl.

Biological Evaluation

The compounds of the invention are found to be active towards δreceptors in warm-blooded animal, e.g., human. Particularly thecompounds of the invention are found to be effective δ receptor ligands.In vitro assays, infra, demonstrate these surprising activities,especially with regard to agonists potency and efficacy as demonstratedin the rat brain functional assay and/or the human δ receptor functionalassay (low). This feature may be related to in vivo activity and may notbe linearly correlated with binding affinity. In these in vitro assays,a compound is tested for their activity toward δ receptors and IC₅₀ isobtained to determine the selective activity for a particular compoundtowards δ receptors. In the current context, IC₅₀ generally refers tothe concentration of the compound at which 50% displacement of astandard radioactive δ receptor ligand has been observed.

The activities of the compound towards κ and μ receptors are alsomeasured in a similar assay.

In Vitro Model

Cell Culture

-   -   A. Human 293S cells expressing cloned human κ, δ and μ receptors        and neomycin resistance are grown in suspension at 37° C. and 5%        CO₂ in shaker flasks containing calcium-free DMEM10% FBS, 5%        BCS, 0.1% Pluronic F-68, and 600 μg/ml geneticin.    -   B. Rat brains are weighed and rinsed in ice-cold PBS (containing        2.5 mM EDTA, pH 7.4). The brains are homogenized with a polytron        for 30 sec (rat) in ice-cold lysis buffer (50 mM Tris, pH 7.0,        2.5 mM EDTA, with phenylmethylsulfonyl fluoride added just prior        use to 0.5 MmM from a 0.5M stock in DMSO:ethanol).        Membrane Preparation

Cells are pelleted and resuspended in lysis buffer (50 mM Tris, pH 7.0,2.5 mM EDTA, with PMSF added just prior to use to 0.1 mM from a 0.1 Mstock in ethanol), incubated on ice for 15 min, then homogenized with apolytron for 30 sec. The suspension is spun at 1000 g (max) for 10 minat 4° C. The supernatant is saved on ice and the pellets resuspended andspun as before. The supernatants from both spins are combined and spunat 46,000 g(max) for 30 min. The pellets are resuspended in cold Trisbuffer (50 mM Tris/Cl, pH 7.0) and spun again. The final pellets areresuspended in membrane buffer (50 mM Tris, 0.32 M sucrose, pH 7.0).Aliquots (1 ml) in polypropylene tubes are frozen in dry ice/ethanol andstored at −70° C. until use. The protein concentrations are determinedby a modified Lowry assay with sodium dodecyl sulfate.

Binding Assays

Membranes are thawed at 37° C., cooled on ice, passed 3 times through a25-gauge needle, and diluted into binding buffer (50 mM Tris, 3 mMMgCl₂, 1 mg/ml BSA (Sigma A-7888), pH 7.4, which is stored at 4° C.after filtration through a 0.22 m filter, and to which has been freshlyadded 5 μg/ml aprotinin, 10 μM bestatin, 10 μM diprotin A, no DTT).Aliquots of 100 μl are added to iced 12×75 mm polypropylene tubescontaining 100 μl of the appropriate radioligand and 100 μl of testcompound at various concentrations. Total (TB) and nonspecific (NS)binding are determined in the absence and presence of 10 μM naloxonerespectively. The tubes are vortexed and incubated at 25° C. for 60-75min, after which time the contents are rapidly vacuum-filtered andwashed with about 12 ml/tube iced wash buffer (50 mM Tris, pH 7.0, 3 mMMgCl₂) through GF/B filters (Whatman) presoaked for at least 2 h in 0.1%polyethyleneimine. The radioactivity (dpm) retained on the filters ismeasured with a beta counter after soaking the filters for at least 12 hin minivials containing 6-7 ml scintillation fluid. If the assay is setup in 96-place deep well plates, the filtration is over 96-placePEI-soaked unifilters, which are washed with 3×1 ml wash buffer, anddried in an oven at 55° C. for 2 h. The filter plates are counted in aTopCount (Packard) after adding 50 μl MS-20 scintillation fluid/well.

Functional Assays

The agonist activity of the compounds is measured by determining thedegree to which the compounds receptor complex activates the binding ofGTP to G-proteins to which the receptors are coupled. In the GTP bindingassay, GTP[γ]³⁵S is combined with test compounds and membranes fromHEK-293S cells expressing the cloned human opioid receptors or fromhomogenised rat and mouse brain. Agonists stimulate GTP[γ]³⁵S binding inthese membranes. The EC₅₀ and E_(max) values of compounds are determinedfrom dose-response curves. Right shifts of the dose response curve bythe delta antagonist naltrindole are performed to verify that agonistactivity is mediated through delta receptors. For human δ receptorfunctional assays, EC₅₀ (low) is measured when the human δ receptorsused in the assay were expressed at lower levels in comparison withthose used in determining EC₅₀ (high). The E_(max) values weredetermined in relation to the standard δ agonist SNC80, i.e., higherthan 100% is a compound that have better efficacy than SNC80.

Procedure for Rat Brain GTP

Rat brain membranes are thawed at 37° C., passed 3 times through a25-gauge blunt-end needle and diluted in the GTPγS binding (50 mM Hepes,20 mM NaOH, 100 mM NaCl, 1 mM EDTA, 5 mM MgCl₂, pH 7.4, Add fresh: 1 mMDTT, 0.1% BSA). 120 μM GDP final is added membranes dilutions. The EC50and Emax of compounds are evaluated from 10-point dose-response curvesdone in 300 μl with the appropriate amount of membrane protein (20μg/well) and 100000-130000 dpm of GTPγ³⁵S per well (0.11-0.14 nM). Thebasal and maximal stimulated binding are determined in absence andpresence of 3 μM SNC-80

Data Analysis

The specific binding (SB) was calculated as TB-NS, and the SB in thepresence of various test compounds was expressed as percentage ofcontrol SB. Values of IC₅₀ and Hill coefficient (n_(H)) for ligands indisplacing specifically bound radioligand were calculated from logitplots or curve fitting programs such as Ligand, GraphPad Prism,SigmaPlot, or ReceptorFit. Values of K_(i) were calculated from theCheng-Prussoff equation. Mean ±S.E.M. values of IC₅₀, K_(i) and n_(H)were reported for ligands tested in at least three displacement curves.

Based on the above testing protocols, we find that the compounds of thepresent invention and some of the intermediates used in the preparationthereof are active toward human δ receptors. Biological activity of thecompounds and selected intermediates of the present invention isindicated in Tables 1 and 2.

TABLE 1 Human δ (nM) RAT BRAIN EC₅₀ % EMax Human κ Human μ (nM) Compd. #IC₅₀ (high) (high) IC₅₀ IC₅₀ EC₅₀ % EMax 1-11 0.25-0.74 0.55-1.93 89-102247-1636 93-1100 0.93-16.7 135-170

TABLE 2 Human δ (nM) % EMax Human κ Human μ Compd. # IC₅₀ EC₅₀ (low)(low) IC₅₀ IC₅₀ 12-23, 0.18-4.18 3.26-484 58-106 1277-8728 92-6560 andInterm. 4aReceptor Saturation Experiments

Radioligand K_(δ) values are determined by performing the binding assayson cell membranes with the appropriate radioligands at concentrationsranging from 0.2 to 5 times the estimated K_(δ) (up to 10 times ifamounts of radioligand required are feasible). The specific radioligandbinding is expressed as pmole/mg membrane protein. Values of K_(δ) andB_(max) from individual experiments are obtained from nonlinear fits ofspecifically bound (B) vs. nM free (F) radioligand from individualaccording to a one-site model.

Determination of Mechano-Allodynia Using Von Frey Testing

Testing is performed between 08:00 and 16:00 h using the methoddescribed by Chaplan et al. (1994). Rats are placed in Plexiglas cageson top of a wire mesh bottom which allows access to the paw, and areleft to habituate for 10-15 min. The area tested is the mid-plantar lefthind paw, avoiding the less sensitive foot pads. The paw is touched witha series of 8 Von Frey hairs with logarithmically incremental stiffness(0.41, 0.69, 1.20, 2.04, 3.63, 5.50, 8.51, and 15.14 grams; Stoelting,Ill., USA). The von Frey hair is applied from underneath the mesh floorperpendicular to the plantar surface with sufficient force to cause aslight buckling against the paw, and held for approximately 6-8 seconds.A positive response is noted if the paw is sharply withdrawn. Flinchingimmediately upon removal of the hair is also considered a positiveresponse. Ambulation is considered an ambiguous response, and in suchcases the stimulus is repeated.

Testing Protocol

The animals are tested on postoperative day 1 for the FCA-treated group.The 50% withdrawal threshold is determined using the up-down method ofDixon (1980). Testing is started with the 2.04 g hair, in the middle ofthe series. Stimuli are always presented in a consecutive way, whetherascending or descending. In the absence of a paw withdrawal response tothe initially selected hair, a stronger stimulus is presented; in theevent of paw withdrawal, the next weaker stimulus is chosen. Optimalthreshold calculation by this method requires 6 responses in theimmediate vicinity of the 50% threshold, and counting of these 6responses begins when the first change in response occurs, e.g. thethreshold is first crossed. In cases where thresholds fall outside therange of stimuli, values of 15.14 (normal sensitivity) or 0.41(maximally allodynic) are respectively assigned. The resulting patternof positive and negative responses is tabulated using the convention,X=no withdrawal; O=withdrawal, and the 50% withdrawal threshold isinterpolated using the formula:50% g threshold=10^((Xf+kδ))/10,000where Xf=value of the last von Frey hair used (log units); k=tabularvalue (from Chaplan et al. (1994)) for the pattern of positive/negativeresponses; and δ=mean difference between stimuli (log units). Hereδ=0.224.

Von Frey thresholds are converted to percent of maximum possible effect(% MPE), according to Chaplan et al. 1994. The following equation isused to compute % MPE:

${\%\mspace{14mu}{MPE}} = {\frac{\begin{matrix}{{{Drug}\mspace{14mu}{treated}\mspace{14mu}{threshold}\mspace{14mu}(g)} -} \\{{allodynia}\mspace{14mu}{threshold}\mspace{14mu}(g)}\end{matrix}}{\begin{matrix}{{{{Control}\mspace{14mu}{threshold}\mspace{14mu}(g)} -}\mspace{14mu}} \\{{allodynia}\mspace{14mu}{threshold}\mspace{14mu}(g)}\end{matrix}} \times 100}$Administration of Test Substance

Rats are injected (subcutaneously, intraperitoneally, intravenously ororally) with a test substance prior to von Frey testing, the timebetween administration of test compound and the von Frey test variesdepending upon the nature of the test compound.

Writhing Test

Acetic acid will bring abdominal contractions when administeredintraperitoneally in mice. These will then extend their body in atypical pattern. When analgesic drugs are administered, this describedmovement is less frequently observed and the drug selected as apotential good candidate.

A complete and typical Writhing reflex is considered only when thefollowing elements are present: the animal is not in movement; the lowerback is slightly depressed; the plantar aspect of both paws isobservable. In this assay, compounds of the present inventiondemonstrate significant inhibition of writhing responses after oraldosing of 1-100 μmol/kg.

(i) Solutions Preparation

-   Acetic acid (AcOH): 120 μL of Acetic Acid is added to 19.88 ml of    distilled water in order to obtain a final volume of 20 ml with a    final concentration of 0.6% AcOH. The solution is then mixed    (vortex) and ready for injection.-   Compound (drug): Each compound is prepared and dissolved in the most    suitable vehicle according to standard procedures.    (ii) Solutions Administration

The compound (drug) is administered orally, intraperitoneally (i.p.),subcutaneously (s.c.) or intravenously (i.v.)) at 10 ml/kg (consideringthe average mice body weight) 20, 30 or 40 minutes (according to theclass of compound and its characteristics) prior to testing. When thecompound is delivered centrally: Intraventricularly (i.c.v.) orintrathecally (i.t.) a volume of 5 μL is administered.

The AcOH is administered intraperitoneally (i.p.) in two sites at 10ml/kg (considering the average mice body weight) immediately prior totesting.

(iii) Testing

The animal (mouse) is observed for a period of 20 minutes and the numberof occasions (Writhing reflex) noted and compiled at the end of theexperiment. Mice are kept in individual “shoe box” cages with contactbedding. A total of 4 mice are usually observed at the same time: onecontrol and three doses of drug.

For the anxiety and anxiety-like indications, efficacy has beenestablished in the geller-seifter conflict test in the rat.

For the functional gastrointestina disorder indication, efficacy can beestablished in the assay described by Coutinho S V et al., in AmericanJournal of Physiology—Gastrointestinal & Liver Physiology.282(2):G307-16, 2002 February, in the rat.

Additional In Vivo Testing Protocols

Subjects and Housing

Naïve male Sprague Dawley rats (175-200 g) are housed in groups of 5 ina temperature controlled room (22° C., 40-70% humidity, 12-hlight/dark). Experiments are performed during the light phase of thecycle. Animals have food and water ad libitum and are sacrificedimmediately after data acquisition.

Sample

Compound (Drug) testing includes groups of rats that do not receive anytreatment and others that are treated with E. colilipopolysaccharide(LPS). For the LPS-treated experiment, four groups areinjected with LPS, one of the four groups is then vehicle-treated whilstthe other three groups are injected with the drug and its vehicle. Asecond set of experiments are conducted involving five groups of rats;all of which receive no LPS treatment. The naïve group receives nocompound (drug) or vehicle; the other four groups are treated withvehicle with or without drug. These are performed to determineanxiolytic or sedative effects of drugs which can contribute to areduction in USV.

Administration of LPS

Rats are allowed to habituate in the experimental laboratory for 15-20min prior to treatment. Inflammation is induced by administration of LPS(endotoxin of gram-negative E. coli bacteria serotype 0111:B4, Sigma).LPS (2.4 μg) is injected intracerebro-ventricularly (i.c.v.), in avolume of 10 μl, using standard stereotaxic surgical techniques underisoflurane anaesthesia. The skin between the ears is pushed rostrallyand a longitudinal incision of about 1 cm is made to expose the skullsurface. The puncture site is determined by the coordinates: 0.8 mmposterior to the bregma, 1.5 mm lateral (left) to the lambda (sagittalsuture), and 5 mm below the surface of the skull (vertical) in thelateral ventricle. LPS is injected via a sterile stainless steel needle(26-G ⅜) of 5 mm long attached to a 100-μl Hamilton syringe bypolyethylene tubing (PE20; 10-15 cm). A 4 mm stopper made from a cutneedle (20-G) is placed over and secured to the 26-G needle by siliconeglue to create the desired 5 mm depth.

Following the injection of LPS, the needle remains in place for anadditional 10 s to allow diffusion of the compound, then is removed. Theincision is closed, and the rat is returned to its original cage andallowed to rest for a minimum of 3.5 h prior to testing.

Experimental Setup for Air-puff Stimulation

The rats remains in the experimental laboratory following LPS injectionand compound (drug) administration. At the time of testing all rats areremoved and placed outside the laboratory. One rat at a time is broughtinto the testing laboratory and placed in a clear box (9×9×18 cm) whichis then placed in a sound-attenuating ventilated cubicle measuring62(w)×35(d)×46(h) cm (BRS/LVE, Div. Tech-Serv Inc). The delivery ofair-puffs, through an air output nozzle of 0.32 cm, is controlled by asystem (AirStim, San Diego Intruments) capable of delivering puffs ofair of fixed duration (0.2 s) and fixed intensity with a frequency of 1puff per 10 s. A maximum of 10 puffs are administered, or untilvocalisation starts, which ever comes first. The first air puff marksthe start of recording.

Experimental Setup for and Ultrasound Recording

The vocalisations are recorded for 10 minutes using microphones(G.R.A.S. sound and vibrations, Vedbaek, Denmark) placed inside eachcubicle and controlled by LMS (LMS CADA-X 3.5B, Data AcquisitionMonitor, Troy, Mich.) software. The frequencies between 0 and 32000 Hzare recorded, saved and analysed by the same software (LMS CADA-X 3.5B,Time Data Processing Monitor and UPA (User Programming and Analysis)).

Compounds (Drugs)

All compounds (drugs) are pH-adjusted between 6.5 and 7.5 andadministered at a volume of 4 ml/kg. Following compound (drug)administration, animals are returned to their original cages until timeof testing.

Analysis

The recording is run through a series of statistical and Fourieranalyses to filter (between 20-24 kHz) and to calculate the parametersof interest. The data are expressed as the mean±SEM. Statisticalsignificance is assessed using T-test for comparison between naive andLPS-treated rats, and one way ANOVA followed by Dunnett's multiplecomparison test (post-hoc) for drug effectiveness. A difference betweengroups is considered significant with a minimum p value of ≦0.05.Experiments are repeated a minimum of two times.

EXAMPLES

The invention will further be described in more detail by the followingExamples which describe methods whereby compounds of the presentinvention may be prepared, purified, analyzed and biologically tested,and which are not to be construed as limiting the invention.

Intermediate 1 N,N-Diethyl-4-formylbenzamide

To a suspension of 4-carboxybenzaldehyde (30 g, 0.2 mole) in 100 ml oftoluene was added SOCl₂(97 ml, 1.3 moles) at 60° C. The reaction washeated until gas evolution ceased followed by evaporation to drynesswith toluene (3×50 mL) This yielded a residue, which was dissolved inCH₂Cl₂ (200 mL). To this solution, cooled in an ice bath while stirring,was added diethylamine (50 mL). Stirring was continued for one hour andthen the mixture heated at reflux for a further hour. After cooling, themixture washed successively with H₂O, 2 N HCl, H₂O then 2 N NaOH andfinally with H₂O. The solution was dried over MgSO₄, filtered andconcentrated to dryness yielding 41 g of oil. Distillation at 140-150°C./1.5 torr gave 36.9 g, 90% of INTERMEDIATE 1.

Intermediate 2 tert-Butyl4-((3-bromophenyl){4-[(diethylamino)carbonyl]phenyl}methyl)piperazine-1-carboxylate

A solution of INTERMEDIATE 1 (6.84 g, 33.3 mmol) benzotriazole (3.96 g,33.3 mmol) and N-Boc piperidine (6.19 g, 33.3 mmol) in 200 mL of toluenewas heated over night under Dean-Stark conditions. The reaction wasconcentrated, dissolved in 45 ml of dry THF and cooled in an ice bath.To this was cannulated 3-bromophenylzinc iodide (0.5 M in THF, 100 mL,50 mmol) over 15 minutes. The reaction was warmed to room temperaturestirred for 30 minutes then heated overnight at 50° C. The reactionmixture was quenched with NH₄Cl, stirred for 15 minutes then extracted 4times with ethyl acetate. The combined organic layers were dried overMgSO₄, filtered and concentrated. The resulting oil was purified byflash chromatography eluting ethyl acetate/heptane 30/70 to 50/50.Yield: 5.86 g, 33% of INTERMEDIATE 2.

Intermediate 34-[[4-[(diethylamino)carbonyl]phenyl][3-[(methoxycarbonyl)amino]phenyl]methyl]-1-piperazinecarboxylicacid, 1,1-dimethylethyl ester

To a solution of INTERMEDIATE 2 (5.39 g, 10.17 mmol) in dry dioxane,while bubbling N₂, was added methyl carbamate (0.99 g, 13.2 mmol),xantphos (0.47 g, 0.81 mmol), Cs₂CO₃ (4.63 g, 14.2 mmol) and Pd₂(dba)₃(0.465 g, 0.51 mmol). The reaction was heated for 7 hrs at reflux,cooled and filtered through diatomaceous earth. The resulting oil waspurified by flash chromatography 40/60 to 80/20 ethyl acetate/heptane.3.3 g, 62% of INTERMEDIATE 3 was obtained.

Intermediate 4[3-[[4-[(diethylamino)carbonyl]phenyl]-1-piperazinylmethyl]phenyl]-carbamicacid, methyl ester

To a solution of INTERMEDIATE 3 (0.788 g, 1.5 mmol) in 15 mL of CH₂Cl₂at 0° C. was added TFA (1.15 ml, 15 mmol). This was stirred untilcompletion by HPLC. The solution was concentrated, dissolved in 20 mL ofethyl acetate and washed with 10 mL 2 N K₂CO₃. The organic layer wasseparated and the aqueous layer washed with 5×20 ml of ethyl acetate.The combined organic layers were dried over MgSO₄, filtered andconcentrated yielding 570 mg, 89% of INTERMEDIATE 4. The INTERMEDIATE 4was separated by chiral HPLC on Chiralpak AD 20% EtOH/80% Heptaneyielding 200 mg of enantiomer INTERMEDIATE 4a and 185 mg of enantiomerINTERMEDIATE 4b.

-   Analytical Chiral HPLC Conditions:-   Chiralpak AD 15% EtOH/85% Heptane-   Flow rate 1 ml/minute-   INTERMEDIATE 4a retention time 25.7 minutes-   INTERMEDIATE 4b retention time 17.5 minutes

Intermediate 5 4-Iodo-N,N-diethylbenzamide

To a mixture of 4-iodo-benzoyl chloride (75 g) in 500 mL CH₂Cl₂ wasadded a mixture of Et₃N (50 mL) and Et₂NH (100 mL) at 0° C. After theaddition, the resulting reaction mixture was warmed up to roomtemperature in 1 hr and was then washed with saturated ammoniumchloride. The organic extract was dried (Na₂SO₄), filtered andconcentrated. Residue was recrystallized from hot hexanes to give 80 gof INTERMEDIATE 5.

Intermediate 6 4-[hydroxy(3-nitrophenyl)methyl]-N,N-diethylbenzamide

INTERMEDIATE 5, N,N-Diethyl-4-iodobenzamide (5.0 g, 16 mmol), wasdissolved in THF (150 mL) and cooled to −78° C. under nitrogenatmosphere. n-BuLi (15 mL, 1.07 M solution in hexane, 16 mmol) was addeddropwise during 10 min at −65 to −78° C. The solution was thencannulated into 3-nitrobenzaldehyde (2.4 g, 16 mmol) in toluene/THF(approx. 1:1, 100 mL) at −78° C. NH₄Cl (aq.) was added after 30 min.After concentration in vacuo, extraction with EtOAc/water, drying(MgSO₄) and evaporation of the organic phase, the residue was purifiedby chromatography on silica (0-75% EtOAc/heptane) to give INTERMEDIATE 6(2.6 g, 50%). ¹H NMR (CDCl₃) δ 1.0-1.3 (m, 6H), 3.2, 3.5 (2 m, 4H), 5.90(s, 1H), 7.30-7.40 (m, 4H), 7.50 (m, 1H), 7.70 (d, J=8 Hz, 1H), 8.12 (m,1H), 8.28 (m, 1H).

Intermediate 7 RacemicN,N-diethyl-4-[(3-nitrophenyl)(1-piperazinyl)methyl]benzamide

To a solution of INTERMEDIATE 6 (10.01 g, 30.5 mmol) in dichloromethane(200 mL) was added thionyl bromide (2.58 mL, 33.6 mmol). After one hourat room temperature the reaction washed with saturated aqueous sodiumbicarbonate (100 mL) and the organic layer was separated. The aqueouslayer was washed with dichloromethane (3×100 mL) and the combinedorganic extracts were dried (Na₂SO₄), filtered and concentrated.

The resulting product in the organic extracts was dissolved inacetonitrile (350 mL) and piperazine (10.5 g, 122 mmol) was added. Afterheating the reaction for one hour at 65° C. the reaction washed withsaturated ammonium chloride/ethyl acetate and the organic layer wasseparated. The aqueous layer was extracted with ethyl acetate (3×100 mL)and the combined organic extracts were dried (Na₂SO₄), filtered andconcentrated to give racemic INTERMEDIATE 7.

Intermediate 8(−)-N,N-diethyl-4-[(3-nitrophenyl)(1-piperazinyl)methyl]benzamide

Racemic INTERMEDIATE 7 is resolved to give enantiomerically pureINTERMEDIATE 8 as follows:

INTERMEDIATE 7 was dissolved in ethanol (150 mL) anddi-p-toluoyl-D-tartaric acid (11.79 g, 1 equivalent) was added. Theproduct precipitated out over a 12 hour period. The solid was collectedby filtration and was redissolved in refluxing ethanol until all of thesolid dissolved (approximately 1200 mL ethanol). Upon cooling the solidwas collected by filtration and the recrystallation repeated a secondtime. The solid was collected by filtration and was treated with aqueoussodium hydroxide (2M) and was extracted with ethyl acetate. The organicextract was then dried (Na₂SO₄), filtered and concentrated to give 1.986g of enantiomerically pure INTERMEDIATE 8. ¹H NMR (CDCl₃) δ 1.11 (br s,3H), 1.25 (br s, 3H), 2.37 (br s, 4H), 2.91 (t, J=5 Hz, 4H), 3.23 (br s,2H), 3.52 (br s, 2H), 4.38 (s, 1H), 7.31-7.33 (m, 2H), 7.41-7.43 (m,2H), 7.47 (t, J=8 Hz, 1H), 7.75-7.79 (m, 1H), 8.06-8.09 (m, 1H),8.30-8.32 (m, 1H).

Chiral purity was determined by HPLC using the following conditions:

-   -   Chiralpack AD column (Daicel Chemical Industries)    -   Flow rate 1 ml/minute    -   Run time 30 minutes at 25° C.    -   Isocratic 15% ethanol 85% hexanes    -   Retention time of molecule=20 minutes        INTERMEDIATE 9 may be obtained by the above procedure but using        di-p-toluoyl-L-tartaric acid in place of di-p-toluoyl-D-tartaric        acid.

Intermediate 10N,N-diethyl-4-[(3-nitrophenyl)[4-(phenylmethyl)-1-piperazinyl]methyl]-benzamide

To a solution of INTERMEDIATE 8 (1.407 g, 3.55 mmol) in1,2-dichloroethane (30 mL) was added benzaldehyde (0.58 mL, 5.71 mmol)and sodium triacetoxyborohydride (1.21 g, 5.71 mmol). After 20 hours atroom temperature the reaction was quenched with aqueous sodiumbicarbonate and the organic layer was separated. The aqueous layer wasextracted with dichloromethane (3×50 mL) and the combined organicextracts were dried (Na₂SO₄), filtered and concentrated. The residue waspurified by flash chromatography, eluting 5% methanol in dichloromethaneto give INTERMEDIATE 10 as a colourless foam (1.576 g, 91% yield).

Intermediate 114-[(3-aminophenyl)[4-(phenylmethyl)-1-piperazinyl]methyl]-N,N-diethyl-benzamide

To a solution of INTERMEDIATE 10 (1.576 g, 3.24 mmol) in a mixture ofethanol, tetrahydrofuran, water and aqueous saturated ammonium chloride(4:2:1:1 ratio v/v) (30 mL) was added granules of iron (1.80 g, 32.4mmol). After 4 hours at reflux (90° C.) the reaction was cooled to roomtemperature, filtered through celite and concentrated. To the residuewas added aqueous sodium bicarbonate and dichloromethane. The organiclayer was separated and the aqueous layer was extracted withdichloromethane (3×50 mL) and the combined organic extracts were dried(Na₂SO₄), filtered and concentrated. The product was purified on silicagel eluting with 1% to 5% methanol in dichloromethane to affordINTERMEDIATE 11 (1.310 g, 88% yield).

Chiral HPLC conditions:

-   -   Chiralpack AD column (Daicel Chemical Industries)    -   Flow rate: 1 ml/minute    -   Run time 30 minutes at 25° C.    -   Isocratic 30% isopropanol/70% hexane    -   Retention time of molecule=18.7 minutes

Intermediate 14 4-{(S)-(3-aminophenyl)[4-(pyridin-3-ylmethyl)piperazin-1-yl]methyl}-N,N-diethylbenzamide

To a solution of INTERMEDIATE 9 (452 mg) in 1,2-dichloroethane (10 ml)was added 3-pyridine carboxaldehyde (215 μL; 2 eq) and sodiumtriacetoxyborohydride (483 mg; 2 eq). The reaction was stirred at roomtemperature under nitrogen for 18 hours and concentrated. Saturatedsodium bicarbonate was added and the aqueous solution was extracted withthree portions of dichloromethane and the combined organics were driedover anhydrous sodium sulfate, filtered and concentrated to yieldINTERMEDIATE 12. The crude INTERMEDIATE 12 was dissolved in a mixture ofethanol, tetrahydrofuran, water and saturated ammonium chloride (4 ml;ratios 4:2:1:1 v/v). Iron nanoparticules (3 tips of spatula) were addedand the solution was heated at 150° C. for 10 minutes in the microwave.The resulting mixture was cooled, filtered through celite andconcentrated. The residue was purified by flash chromatography on silicagel, eluting with a gradient from 1% to 10% MeOH in dichloromethane togive INTERMEDIATE 14 (312 mg, 60% yield) as a colourless solid.

Intermediate 15 4-{(S)-(3-aminophenyl)[4-(1,3-thiazol-2-ylmethyl)piperazin-1-yl]methyl}-N,N-diethylbenzamide

To a solution of INTERMEDIATE 9 (479 mg) in 1,2-dichloroethane (13 ml)was added 2-thiazole carboxaldehyde (212 μL; 2 eq) and sodiumtriacetoxyborohydride (510 mg; 2 eq). The reaction was stirred at roomtemperature under nitrogen for 18 hours. Saturated sodium bicarbonatewas added and the aqueous solution was extracted with three portions ofdichloromethane and the combined organics were dried over anhydroussodium sulfate, filtered and concentrated to give INTERMEDIATE 13. Thecrude INTERMEDIATE 13 was dissolved in a mixture of ethanol,tetrahydrofuran, water and saturated ammonium chloride (4 ml; ratios4:2:1:1 v/v). Iron nanoparticules (3 tips of spatula) were added and thesolution was heated at 150° C. for 10 minutes in the microwave. Theresulting mixture was cooled, filtered through celite and concentrated.The residue was purified by reverse phase chromatography, eluting 10% to45% acetonitrile in water containing 0.1% trifluoroacetic acid. Theproduct was obtained as the trifluoroacetic acid salt and waslyophilized to give INTERMEDIATE 15 (372 mg, 39% yield) as a colourlesssolid.

Intermediate 16a or 16b tert-Butyl4-((3-aminophenyl){4-[(diethylamino)carbonyl]phenyl}methyl)piperazine-1-carboxylate

To a solution of INTERMEDIATE 8 or 9 (300 mg) in dioxane (40 ml) wasadded di-tert-butyl dicarbonate (247 mg; 1.5 eq). Sodium carbonate (119mg; 1.5 eq) was dissolved in water (15 ml) and then added in the dioxanesolution. After 12 hours the solution was concentrated and saturatedsodium bicarbonate was then added. The aqueous solution was extractedwith three portions of dichloromethane and the combined organics weredried over anhydrous sodium sulfate, filtered and concentrated to afforda white foam. Without further purification, the foam was then dissolvedin a mixture of ethanol, tetrahydrofuran, water and saturated ammoniumchloride (15 ml; ratios 4:2:1:1 v/v). Iron granules (422 mg; 10 eq) wereadded and the solution was heated at 90° C. for 1.5 hour. The resultingmixture was cooled, filtered through celite and concentrated. Saturatedsodium bicarbonate was added and the aqueous solution was extracted withthree portions of dichloromethane and the combined organics were driedover anhydrous sodium sulfate, filtered and concentrated to afford awhite foam INTERMEDIATE 16a or 16b, respectively. The product can be usewithout any further purification. (92-99% yield). ¹H NMR (400 MHz,CDCl₃) 1.06-1.16 (m, 3H), 1.17-1.26 (m, 3H), 1.44 (s, 9H), 2.28-2.39 (m,4H), 3.20-3.31 (br s, 2H), 3.37-3.44 (br s, 2H), 3.48-3.58 (br s, 2H),3.60-3.70 (br s, 2H), 4.12 (s, 1H), 6.51-6.55 (m, 1H), 6.72 (t, J=2.13Hz, 1H), 6.79 (d, J=8.17 Hz, 1H), 7.06 (t, J=7.46 Hz, 1H), 7.29 (d,J=7.82 Hz, 2H), 7.43 (d, J=7.82 Hz, 2H).

Compound 1 R-Methyl3-[(4-[(diethylamino)carbonyl]phenyl)(4-benzyl-piperazin-1-yl)methyl]phenylcarbamate

To a solution of INTERMEDIATE 4a (200 mg, 0.47 mmol) dissolved in 5 mldichloroethane was added benzaldehyde (95.5 μL, 0.94 mmol) andNaBH(OAc)₃ (200 mg, 0.94 mmol). The reaction was stirred at roomtemperature overnight. Then 5 ml of a saturated solution of NaHCO₃ wasadded and the aqueous layer extracted 4 times with CH₂Cl₂. The combinedorganic layers were dried over MgSO₄, filtered and concentrated.Purification by reverse phase chromatography yielded 188 mg of COMPOUND1 under the conditions: LUNA C-18, gradient 10-50% B in 25 min, flow: 40mL/min, 20° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN. ¹H NMR (400MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m, 3H), 2.31 (m, 2H), 3.04 (m, 2H),3.24 (m, 4H), 3.39 (m, 2H), 3.51 (m, 2H), 3.72 (s, 3H), 4.34 (s, 2H),4.43 (s, 1H), 7.09 (m, 1H), 7.20 (m, 2H), 7.32 (d, J=8.2 Hz, 2 H), 7.48(s, 5H), 7.55 (d, J=8.2 Hz, 2 H), 7.70 (s, 1H). Anal. calcd forC₃₁H₃₈N₄O₃×2.10 C₂HF₃O₂×0.3H₂O: C, 55.66; H, 5.40; N, 7.38. Found: C,55.70; H, 5.24; N, 7.41. M.S (calcd): 515.30 (MH+), M.S (found): 515.55(MH+). HPLC: k′: 2.95; Purity: >99% (215 nm), >99% (254 nm), >99% (280nm). Conditions: Zorbax C-18, gradient 30-80% B in 25 min, flow: 1mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >90% (215 nm), >94% (254 nm), >94% (280 nm), Rt: 8.7 mins;Conditions: Chiralpak AD 30% IPA/70% hexane. Rotation: [α]¹⁷ _(D)=−14.5°(c=0.74, EtOH).

Compound 1R-[3-[[4-[(diethylamino)carbonyl]phenyl][4-(phenylmethyl)-1-piperazinyl]methyl]phenyl]-carbamicacid methyl ester (via another synthetic route)

Methyl chloroformate (0.008 mL, 0.11 mmol) and zinc dust (8 mg, 0.11mmol) were stirred together in 1 mL of dry toluene at room temperatureunder nitrogen for 10 minutes. A 1 mL toluene solution of INTERMEDIATE10 (50 mg, 0.110 mmol) was then added dropwise and the reaction mixturewas stirred at room temperature overnight. The solution was then dilutedwith dichloromethane and filtered. The organic phase washed withsaturated NaHCO₃ solution, brine and dried over anhydrous Na₂SO₄. Theproduct was purified by silica gel flash chromatography eluting 50%hexanes in acetone to afford COMPOUND 1 (28 mg, 50% yield). ¹H NMR (400MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m, 3H), 2.31 (m, 2H), 3.04 (m, 2H),3.24 (m, 4H), 3.39 (m, 2H), 3.51 (m, 2H), 3.72 (s, 3H), 4.34 (s, 2H),4.43 (s, 1H), 7.09 (m, 1H), 7.20 (m, 2H), 7.32 (d, J=8.2 Hz, 2 H), 7.48(s, 5H), 7.55 (d, J=8.2 Hz, 2 H), 7.70 (s, 1H). Anal. calcd forC₃₁H₃₈N₄O₃×2.10 C₂HF₃O₂×0.3H₂O: C, 55.66; H, 5.40; N, 7.38. Found: C,55.70; H, 5.24; N, 7.41. M.S (calcd): 515.30 (MH+), M.S (found): 515.55(MH+). HPLC: k′: 2.95; Purity: >99% (215 nm), >99% (254 nm), >99% (280nm). Conditions: Zorbax C-18, gradient 30-80% B in 25 min, flow: 1mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >90% (215 nm)>94% (254 nm)>94% (280 nm), Retention time: 8.7mins; Conditions: Chiralpak AD 30% IPA/70% hexane. Rotation: [α]¹⁷_(D)=−14.5° (c=0.74, EtOH).

Compound 2 S-Methyl3-[(4-[(diethylamino)carbonyl]phenyl)(4-benzyl-piperazin-1-yl)methyl]phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (185mg, 0.43 mmol) and benzaldehyde (100 □L, 0.87 mmol) afforded 178 mg ofCOMPOUND 2. ¹H NMR (400 MHz, CD₃OD): δ 1.09 (m, 3H), 1.22 (m, 3H), 2.31(m, 2H), 3.04 (m, 2H), 3.24 (m, 4H), 3.40 (m, 2H), 3.51 (m, 2H), 3.72(s, 3H), 4.34 (s, 2H), 4.43 (s, 1H), 7.09 (m, 1H), 7.19 (m, 2H), 7.32(d, J=8.2 Hz, 2 H), 7.48 (s, 5H), 7.55 (d, J=8.2 Hz, 2 H), 7.71 (s, 1H).Anal. calcd for C₃₁H₃₈N₄O₃×1.60 C₂HF₃O₂×0.6H₂O: C, 58.47; H, 5.77 N,7.98. Found: C, 58.50; H, 5.70; N, 8.06. M.S (calcd): 515.30 (MH⁺), M.S(found): 515.57 (MH⁺). HPLC: k′: 3.00; Purity: >99% (215 nm), >99% (254nm), >99% (280 nm). HPLC Conditions: Zorbax C-18, gradient 30-80% B in25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA inCH₃CN; Chiral Purity: >99% (215 nm)>99% (254 nm)>99% (280 nm), Rt: 11.2min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation:[α]¹⁷ _(D)=+17.1° (c=0.77, EtOH).

Compound 3 S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 2-thiophenecarboxaldehyde (66 □L, 0.70 mmol) afforded213 mg of COMPOUND 3. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m,3H), 2.35 (br s, 2H), 3.04 (br s, 2H), 3.32 (m, 6H), 3.51 (m, 2H), 3.72(s, 3H), 4.44 (s, 1H), 4.58 (s, 2H), 7.09 (m, 1H), 7.13 (dd, J=3.7,J=5.1 Hz, 3H), 7.21 (m, 2 H), 7.32 (m, 3 H), 7.55 (d, J=8.2 Hz, 2 H),7.62 (m, 1 H), 7.69 (br s, 1H). Anal. calcd for C₂₉H₃₆N₄O₃S×1.40C₂HF₃O₂×0.9H₂O: C, 54.84; H, 5.67; N, 8.04. Found: C, 54.76; H, 5.65; N,8.09. M.S (calcd): 521.26 (MH⁺), M.S (found): 521.26 (MH⁺). HPLC: k′:6.51; Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm). HPLCConditions: Zorbax C-18, gradient 30-80% B in 25 min, flow: 1 mL/min,25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; Chiral Purity: >97%(215 nm)>96% (254 nm)>96% (280 nm), Rt: 22.2 min; Chiral HPLCConditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁷_(D)=+11.3° (c=1.14, MeOH).

Compound 4 R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 2-thiophenecarboxaldehyde (66 □L, 0.70 mmol) afforded197 mg of COMPOUND 4. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m,3H), 2.35 (br s, 2H), 3.02 (br s, 2H), 3.32 (m, 6H), 3.51 (m, 2H), 3.72(s, 3H), 4.44 (s, 1H), 4.58 (s, 2H), 7.09 (m, 1H), 7.13 (m, 1H), 7.21(m, 2H), 7.32 (m, 3H), 7.55 (d, J=8.0 Hz, 2H), 7.62 (d, J=5.1 Hz, 2H),7.69 (s, 1H). Anal. calcd for C₂₉H₃₆N₄O₃S×1.5 C₂HF₃O₂×0.9H₂O: C, 55.13;H, 5.51; N, 8.04. Found: C, 55.14; H, 5.55; N, 8.11. M.S. (calcd):521.26 (MH⁺), M.S. (found): 521.23 (MH⁺). HPLC: k′: 6.59; Purity: >99%(215 nm), >99% (254 nm), >99% (280 nm). HPLC Conditions: Zorbax C-18,gradient 20-50% B in 25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O,B: 0.1% TFA in CH₃CN; Chiral Purity: >99% (215 nm)>99% (254 nm)>99% (280nm), Retention time: 11.8 min; Chiral HPLC Conditions: Chiralpak AD, 30%IPA/70% hexane. Rotation: [α]¹⁷ _(D)=−12.8° (c=0.96, MeOH).

Compound 5 S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-3-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 3-thiophenecarboxaldehyde (66 □L, 0.70 mmol) afforded205 mg of COMPOUND 5. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m,3H), 2.36 (m, 2H), 2.99 (m, 2H), 3.28 (m, 6H), 3.51 (m, 2H), 3.72 (s,3H), 4.36 (s, 2H), 4.42 (s, 1H), 7.09 (m, 1H), 7.21 (m, 3H), 7.32 (d,J=8.0 Hz, 2 H), 7.55 (m, 3H), 7.67 (m, 2H). Anal. calcd forC₂₉H₃₆N₄O₃S×1.7 C₂HF₃O₂×0.3H₂O: C, 54.05; H, 5.36; N, 7.78. Found: C,54.08; H, 5.36; N, 7.70. M.S. (calcd): 521.26 (MHV), M.S. (found):521.26 (MH⁺). HPLC: k′: 6.68; Purity: >99% (215 nm), >99% (254 nm), >99%(280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in 25 min,flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >99% (215 nm), >99% (254 nm), >99% (280 nm), Retention time:13.3 min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane.Rotation: [α]¹⁷ _(D)=+11.3° (c=1.15, MeOH).

Compound 6 R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-3-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 3-thiophenecarboxaldehyde (66 □L, 0.70 mmol) afforded199 mg of COMPOUND 6. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m,3H), 2.31 (br s, 2H), 3.04 (br s, 2H), 3.24 (m, 4H), 3.37 (m, 2H), 3.51(m, 2H), 3.72 (s, 3H), 4.37 (s, 2H), 4.43 (s, 1H), 7.09 (m, 1H), 7.21(m, 3H), 7.32 (d, J=8.2 Hz, 2H), 7.55 (m, 3 H), 7.68 (m, 2H). Anal.calcd for C₂₉H₃₆N₄O₃S×1.4 C₂HF₃O₂×1.0H₂O: C, 54.69; H, 5.69; N, 8.02.Found: C, 54.74; H, 5.63; N, 8.16. M.S. (calcd): 521.26 (MH⁺), M.S.(found): 521.25 (MH⁺). HPLC: k′: 6.67; Purity: >99% (215 nm), >99% (254nm), >99% (280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA inCH₃CN; Chiral Purity: >99% (215 nm)>99% (254 nm)>99% (280 nm), Retentiontime: 9.0 min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane.Rotation: [α]¹⁷ _(D)=−12.9° (c=1.13, MeOH).

Compound 7 S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(2-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 2-furaldehyde (58 μL, 0.70 mmol) afforded 172 mg ofCOMPOUND 7. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.22 (m, 3H), 2.36(br s, 2H), 3.02 (br s, 2H), 3.31 (m, 6H), 3.51 (m, 2H), 3.72 (s, 3H),4.42 (s, 2H), 4.44 (s, 1H), 6.52 (dd, J=1.9, 3.1 Hz, 1H), 6.71 (d, J=3.3Hz, 1H), 7.09 (m, 1H), 7.20 (m, 2H), 7.32 (d, J=8.4 Hz, 2H), 7.55 (d,J=8.2 Hz, 2H), 7.66 (m, 1H), 7.69 (s, 1H).

Anal. calcd for C₂₉H₃₆N₄O₄×1.5 C₂HF₃O₂×0.6H₂O: C, 55.99; H, 5.68; N,8.16.

Found: C, 56.02; H, 5.74; N, 8.22. M.S. (calcd): 505.28 (MH⁺), M.S.(found): 505.26 (MH⁺). HPLC: k′: 5.92; Purity: >99% (215 nm), >99% (254nm), >99% (280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA inCH₃CN; Chiral Purity: >95% (215 nm), >95% (254 nm), >96% (280 nm), Rt:8.3 min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane.Rotation: [α]¹⁷ _(D)=+14.4° (c=1.06, MeOH).

Compound 8 R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(2-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 2-furaldehyde (58 □L, 0.70 mmol) afforded 50 mg ofCOMPOUND 8. ¹H NMR (400 MHz, CD₃OD): δ 1.09 (m, 3H), 1.22 (m, 3H), 2.32(br s, 2H), 3.06 (br s, 2H), 3.24 (m, 4H), 3.40 (m, 2H), 3.51 (m, 2H),3.72 (s, 3H), 4.42 (s, 2H), 4.44 (s, 1H), 6.53 (dd, J=1.8, 3.1 Hz, 1H),6.71 (d, J=3.3 Hz, 1H), 7.09 (m, 1H), 7.20 (m, 2H), 7.32 (d, J=8.2 Hz,2H), 7.55 (d, J=8.2 Hz, 2H), 7.67 (m, 1H), 7.70 (s, 1H). Anal. calcd forC₂₉H₃₆N₄O₄×1.6 C₂HF₃O₂×0.3H₂O: C, 55.85; H, 5.56; N, 8.09. Found: C,55.76; H, 5.50; N, 8.25. M.S. (calcd): 505.28 (MH⁺), M.S. (found):505.27 (MH⁺). HPLC: k′: 6.00; Purity: >99% (215 nm), >99% (254 nm), >99%(280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in 25 min,flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >99% (215 nm), >99% (254 nm), >99% (280 nm), Retention time: 7.2min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation:[α]¹⁷ _(D)=−13.8° (c=0.97, MeOH).

Compound 9 S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(3-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 3-furaldehyde (58 □L, 0.70 mmol) afforded 167 mg ofCOMPOUND 9. ¹H NMR (400 MHz, CD₃OD): δ 1.09 (m, 3H), 1.22 (m, 3H), 2.32(br s, 2H), 3.06 (br s, 2H), 3.24 (m, 4H), 3.40 (m, 2H), 3.51 (m, 2H),3.72 (s, 3H), 4.42 (s, 2H), 4.44 (s, 1H), 6.53 (dd, J=1.8, 3.1 Hz, 1H),6.71 (d, J=3.3 Hz, 1H), 7.09 (m, 1H), 7.20 (m, 2H), 7.32 (d, J=8.2 Hz,2H), 7.55 (d, J=8.2 Hz, 2H), 7.67 (m, 1H), 7.70 (s, 1H). Anal. calcd forC₂₉H₃₆N₄O₄×2.0 C₂HF₃O₂×0.5H₂O: C, 53.44; H, 5.30; N, 7.55. Found: C,53.42; H, 5.28; N, 7.68. M.S. (calcd): 505.28 (MH⁺), M.S. (found):505.27 (MH⁺). HPLC: k′: 5.99; Purity: >99% (215 nm), >99% (254 nm), >99%(280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in 25 min,flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >97% (215 nm)>97% (254 nm)>97% (280 nm), Rt: 13.8 min; ChiralHPLC Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁷_(D)=+13.9° (c=0.94, MeOH).

Compound 10 R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(3-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 3-furaldehyde (58 μL, 0.70 mmol) afforded 119 mg ofCOMPOUND 10. ¹H NMR (400 MHz, CD₃OD): δ 1.09 (m, 3H), 1.22 (m, 3H), 2.32(br s, 2H), 3.05 (br s, 2H), 3.21 (m, 4H), 3.47 (m, 4H), 3.72 (s, 3H),4.24 (s, 2H), 4.43 (s, 1H), 6.58 (m, 1 H), 7.09 (m, 1H), 7.20 (m, 2H),7.32 (d, J=8.2 Hz, 2 H), 7.55 (d, J=8.2 Hz, 2 H), 7.63 (m, 1H), 7.70 (s,1H), 7.76 (s, 1H). Anal. Calcd for C₂₉H₃₆N₄O₄×1.5 C₂HF₃O₂×0.2H₂O: C,56.58; H, 5.62; N, 8.25. Found: C, 56.50; H, 5.56; N, 8.31. M.S.(calcd): 505.28 (MH⁺), M.S. (found): 505.27 (MH⁺). HPLC: k′: 6.02;Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm). HPLC Conditions:Zorbax C-18, gradient 20-50% B in 25 min, flow: 1 mL/min, 25° C., A:0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; Chiral Purity: >99% (215nm), >99% (254 nm), >99% (280 nm), Retention time: 8.7 min; Chiral HPLCConditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁷_(D)=−14.9° (c=1.10, MeOH).

Compound 11 R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(1H-imidazol-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 2-imidazolecarboxaldehyde (68 mg, 0.70 mmol) afforded141 mg of COMPOUND 11. ¹H NMR (400 MHz, CD₃OD): δ 1.09 (m, 3H), 1.23 (m,3H), 2.82 (br s, 4H), 3.02 (br s, 4H), 3.24 (m, 2H), 3.52 (m, 2H), 3.73(s, 3H), 3.98 (s, 2H), 5.03 (s, 1H), 7.27 (m, 3H), 7.42 (d, J=8.0 Hz, 2H), 7.49 (m, 2H), 7.67 (d, J=8.0 Hz, 2 H), 7.84 (s, 1H). Anal. calcd forC₂₈H₃₆N₆O₃×2.1 C₂HF₃O₂×1.5H₂O: C, 50.16; H, 5.37; N, 10.90. Found: C,50.06; H, 5.27; N, 11.02. M.S. (calcd): 505.29 (MH⁺), M.S. (found):505.28 (MH⁺). HPLC: k′: 2.55; Purity: >98% (215 nm), >97% (254 nm), >97%(280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% B in 25 min,flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; ChiralPurity: >99% (215 nm), >99% (254 nm), >99% (280 nm), Retention time:12.5 min; Chiral HPLC Conditions: Chiralpak AD, 30% IPA/70% hexane.Rotation: [α]¹⁶ _(D)=−3.33° (c=1.08, MeOH).

Compound 12S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(pyridin-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 2-pyridine carboxaldehyde (76 mg, 0.70 mmol) afforded213 mg of COMPOUND 12. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.22 (m,3H), 2.74 (br s, 4H), 3.24 (m, 2H), 3.41 (m, 4H), 3.51 (m, 2H), 3.72 (s,3H), 4.48 (s, 2H), 4.48 (s, 1H), 7.11 (m, 1H), 7.21 (m, 2H), 7.33 (d,J=7.7 Hz, 2 H), 7.43 (m, 1H), 7.48 (m, 1H), 7.57 (d, J=7.7 Hz, 2 H),7.73 (s, 1H), 7.89 (s, 1H), 8.66 (m, 1H). Anal. calcd for C₃₀H₃₇N₅O₃ 1.5C₂HF₃O₂×1.2H₂O: C, 55.96; H, 5.82; N, 10.01. Found: C, 55.93; H, 5.73;N, 10.01. M.S. (calcd): 516.30 (MH⁺), M.S. (found): 516.29 (MH⁺). HPLC:k′: 0.88; Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm). HPLCConditions: Zorbax C-18, gradient 30-80% B in 25 min, flow: 1 mL/min,25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; Chiral Purity: >99%(215 nm), >99% (254 nm), >99% (280 nm), Retention time: 12.9 min;Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁶_(D)=+16.5° (c=1.24, MeOH).

Compound 13R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(pyridin-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 2-pyridine carboxaldehyde (76 mg, 0.70 mmol) afforded187 mg of COMPOUND 13. ¹H NMR (400 MHz, CD₃OD): δ 1.08 (m, 3H), 1.21 (m,3H), 2.75 (br s, 4H), 3.24 (m, 2H), 3.41 (m, 4H), 3.51 (m, 2H), 3.72 (s,3H), 4.48 (s, 2H), 4.48 (s, 1H), 7.11 (m, 1H), 7.20 (m, 2H), 7.33 (d,J=8.0 Hz, 2 H), 7.43 (m, 1H), 7.48 (m, 1H), 7.57 (d, J=8.0 Hz, 2 H),7.73 (s, 1H), 7.89 (s, 1H), 8.66 (m, 1H). Anal. calcd for C₃₀H₃₇N₅O₃×1.7C₂HF₃O₂×0.8H₂O: C, 55.42; H, 5.61; N, 9.67. Found: C, 55.40; H, 5.62; N,9.83. M.S. (calcd): 516.30 (MH⁺), M.S. (found): 516.28 (MH⁺). HPLC: k′:3.02; Purity: >97% (215 nm), >98% (254 nm), >99% (280 nm). HPLCConditions: Zorbax C-18, gradient 20-50% B in 25 min, flow: 1 mL/min,25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; Chiral Purity: >99%(215 nm), >99% (254 nm), >99% (280 nm), Retention time: 11.3 min; ChiralHPLC Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁶_(D)=−15.4° (c=1.01, MeOH).

Compound 14S-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(pyridin-4-ylmethyl)piperazin-1-yl}methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4b (200mg, 0.47 mmol) and 4-pyridine carboxaldehyde (76 mg, 0.70 mmol) afforded217 mg of COMPOUND 14. ¹H NMR (Free Amine, 400 MHz, CDCl₃): δ 1.09 (brs, 3H), 1.21 br s, 3H), 2.43 (m, 8H), 3.24 (br s, 2H), 3.50 (s, 2H),3.51 (br s, 2H), 3.76 (s, 3H), 4.22 (s, 1H), 6.63 (s, 1H), 7.10 (m, 1H),7.22 (m, 4H), 7.28 (d, J=8.2 Hz, 2 H), 7.42 (m, 1H), 7.42 (d, J=8.2 Hz,2 H), 8.52 (s, 2H).

Anal. calcd for C₃₀H₃₇N₅O₃×1.9 C₂HF₃O₂×1.8H₂O: C, 53.09; H, 5.60; N,9.16. Found: C, 53.04; H, 5.60; N, 9.18. M.S. (calcd): 516.30 (MH⁺),M.S. (found): 516.28 (MH⁺). HPLC: k′: 2.69; Purity: >99% (215 nm), >99%(254 nm), >99% (280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% Bin 25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA inCH₃CN; Chiral Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm),Retention time: 12.9 min; Chiral HPLC Conditions: Chiralpak AD, 30%IPA/70% hexane. Rotation: [α]¹⁶ _(D)=+10.3° (c=1.25, MeOH).

Compound 15R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(pyridin-4-ylmethyl)piperazin-1-yl}methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (200mg, 0.47 mmol) and 4-pyridine carboxaldehyde (76 mg, 0.70 mmol) afforded247 mg of COMPOUND 15. ¹H NMR (Free Amine, 400 MHz, CDCl₃): δ 1.09 (brs, 3H), 1.21 br s, 3H), 2.45 (m, 8H), 3.24 (br s, 2H), 3.50 (s, 2H),3.51 (br s, 2H), 3.76 (s, 3H), 4.22 (s, 1H), 6.64 (s, 1H), 7.10 (m, 1H),7.22 (m, 4H), 7.28 (d, J=8.2 Hz, 2 H), 7.42 (m, 1H), 7.42 (d, J=8.2 Hz,2 H), 8.52 (d, J=5.7 Hz, 2 H). Anal. calcd for C₃₀H₃₇N₅O₃×2.6C₂HF₃O₂×1.0H₂O: C, 50.93; H, 5.05; N, 8.44. Found: C, 50.89; H, 5.07; N,8.50. M.S. (calcd): 516.30 (MH⁺), M.S. (found): 516.28 (MH⁺). HPLC: k′:2.69; Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm). HPLCConditions: Zorbax C-18, gradient 20-50% B in 25 min, flow: 1 mL/min,25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA in CH₃CN; Chiral Purity: >99%(215 nm), >99% (254 nm), >99% (280 nm), Retention time: 16.3 min;Conditions: Chiralpak AD, 30% IPA/70% hexane. Rotation: [α]¹⁶ _(D)=−8.1°(c=1.10, MeOH).

Compound 16R-Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-2-ylmethyl)-piperazin-1-yl]methyl}phenylcarbamate

Using the same method as for COMPOUND 1 and using INTERMEDIATE 4a (140mg, 0.33 mmol) and 2-thiazole carboxaldehyde (45 mg, 0.39 mmol) afforded85 mg of COMPOUND 16. ¹H NMR (Free Amine, 400 MHz, CDCl₃): δ 1.09 (br s,3H), 1.21 br s, 3H), 2.44 (br s, 4H), 2.61 (br s, 4H), 3.23 (br s, 2H),3.51 (br s, 2H), 3.76 (s, 3H), 3.88 (s, 2H), 4.23 (s, 1H), 6.61 (s, 1H),7.11 (m, 1H), 7.23 (m, 3H), 7.28 (d, J=8.2 Hz, 2 H), 7.41 (m, 1H), 7.43(d, J=8.2 Hz, 2H) 7.70 (d, J=3.3 Hz, 1 H). M.S. (calcd): 522.3 (MH⁺),M.S. (found): 522.2 (MH⁺) HPLC: k′: 4.09; Purity: >99% (215 nm), >99%(254 nm), >99% (280 nm). HPLC Conditions: Zorbax C-18, gradient 20-50% Bin 25 min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B: 0.1% TFA inCH₃CN; Chiral Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm),Retention time: 9.5 min; Chiral HPLC Conditions: Chiralpak AD, 30%IPA/70% hexane. Rotation: [α]¹⁶ _(D)=−12.08° (c=1.01, MeOH).

Compound 17[3-[[4-[(diethylamino)carbonyl]phenyl][4-(phenylmethyl)-1-piperazinyl]methyl]phenyl]methyl-carbamicacid, methyl ester

INTERMEDIATE 3 was methylated with sodium hydride/methyl iodide and theBoc group cleaved with TFA. The secondary amine was reacted withbenzaldehyde and sodium triacetoxyborohydride to give racemic COMPOUND17. This material was purified by chiral HPLC on Chiralpak AD 25%EtOH/75% Heptane to yield enantiomerically pure COMPOUND 17. ¹H NMR (400MHz, CD₃OD): □ 1.09 (m, 3H), 1.22 (m, 3H), 2.31 (m, 2H), 3.05 (m, 2H),3.25 (m, 4H), 3.25 (s, 3H), 3.40 (m, 2H), 3.51 (m, 2H), 3.83 (s, 3H),4.34 (s, 2H), 4.50 (s, 1H), 7.15 (m, 1H), 7.32 (m, 4H), 7.43 (s, 1H),7.49 (s, 5H), 7.55 (d, J=8.2 Hz, 2 H). M.S. (calc'd): 529.3 (MH⁺), M.S.(found): 529.2 (MH⁺). HPLC: k′: 2.42; Purity: >99% (215 nm), >99% (254nm), >99% (280 nm). Conditions: Zorbax C-18, gradient 20-50% B in 25min, flow: 1 mL/min, 25° C., A: 0.1% TFA in H₂O, B:0.1% TFA in CH₃CN;Chiral Purity: >99% (215 nm)>98% (254 nm)>99% (280 nm), Retention time:7.01 min; Conditions: Chiralpak AD, 30% IPA/70% hexane. [α]¹⁶_(D)=−15.69° (c=1.06, MeOH).

Compound 18[3-[(S)-[4-[(diethylamino)carbonyl]phenyl][4-(3-pyridinylmethyl)-1-piperazinyl]methyl]phenyl]-carbamic acid, methyl ester

Methyl chloroformate (0.042 mL, 0.54 mmol) and zinc dust (35 mg, 0.54mmol) were stirred together in 3 mL of dry toluene at room temperatureunder nitrogen for 10 minutes. A 8 mL toluene solution of INTERMEDIATE14 (247 mg, 0.54 mmol) was then added dropwise and the reaction mixturewas stirred at room temperature for one hour. The solution was thendiluted with dichloromethane and filtered. The organic phase washed withsaturated NaHCO₃ solution, brine and dried over anhydrous Na₂SO₄. Theproduct was purified by silica gel flash chromatography eluting 30%hexanes in acetone rising to 27% hexanes, 3% methanol in acetone toafford COMPOUND 18. ¹H NMR (400 MHz, CDCl₃) δ1.09 (br s, 3H), 1.21 (brs, 3H), 2.31-2.54 (m, 8H), 3.23 (br s, 2H), 3.47-3.56 (m, 4H), 3.76 (s,3H), 4.21 (s, 1H), 6.68 (br s, 1H), 7.08 (dt, J=7.21 Hz 1.61 Hz, 1H),7.18-7.25 (m, 3H), 7.27 (d, J=7.86 Hz, 2H), 7.41 (d, J=8.02 Hz, 2H),7.64 (dt, J=7.69 Hz 1.92 Hz, 1H), 8.49 (dd, J=4.97 Hz, 1.60 Hz, 1H),8.52 (d, J=1.76 Hz, 1H). Found: C, 56.47; H, 6.76; N, 10.71.C₃₀H₃₇N₅O₃×1.0H₂O×2.9 HCl×0.2C₄H₁₀O has C, 56.55; H, 6.76; N, 10.70%.M.S. (calc'd): 516.3 (MH⁺), M.S. (found): 516.2 (MH⁺). HPLC: k′: 4.27;Purity: >99% (215 nm), >99% (254 nm), >99% (280 nm). Conditions: ZorbaxC-18, gradient 10-50% B in 25 min, flow: 1 mL/min, 25° C., A: 0.1% TFAin H₂O, B:0.1% TFA in CH₃CN; Chiral Purity: >99% (215 nm)>98% (254nm)>99% (280 nm), Retention time: 6.66 min; Chiral HPLC Conditions:Chiralpak AD, 50% ethanol/50% hexane. Rotation: [α]_(D)=+7.64° (c=0.497,MeOH).

Compound 19[3-[(S)-[4-[(diethylamino)carbonyl]phenyl][4-(2-thiazolylmethyl)-1-piperazinyl]methyl]phenyl]-carbamic acid, methyl ester

Methyl chloroformate (0.031 mL, 0.40 mmol) and zinc dust (26 mg, 0.40mmol) were stirred together in 2 mL of dry toluene at room temperatureunder nitrogen for 10 minutes. A 5 mL toluene solution of INTERMEDIATE15 (185 mg, 0.40 mmol) was then added dropwise and the reaction mixturewas stirred at room temperature for one hour. The solution was thendiluted with dichloromethane and filtered. The organic phase washed withsaturated NaHCO₃ solution, brine and dried over anhydrous Na₂SO₄. Theproduct was purified by silica gel flash chromatography eluting 30%hexanes in acetone rising to 27% hexanes, 3% methanol in acetone toafford COMPOUND 19. ¹HNMR (400 MHz, CD₃OD) δ 1.06 (t, J=6.49 Hz, 3H),1.19 (t, J=6.93 Hz, 3H), 2.90-3.10 (m, 2H), 3.17-3.25 (m, 2H), 3.33-3.42(m, 4H), 3.45-3.52 (m, 2H), 3.70 (s, 3H), 4.59 (s, 2H), 7.16-7.23 (m,1H), 7.26 (d, J=4.00 Hz, 2H), 7.36 (d, J=7.81 Hz, 2H), 7.65 (d, J=7.32Hz, 2H), 7.73-7.8 (m, 2H), 7.88-7.93 (m, 1H).

Found: C, 56.81; H, 6.53; N, 11.56. C₂₈H₃₅N₅O₃S×0.90H₂O 1.5HCl has C,56.75; H, 6.51; N, 11.82%. M.S. (calc'd): 522.3 (MH⁺), M.S. (found):522.2 (MH⁺). HPLC: k′: 3.99; Purity: >99% (215 nm), >99% (254 nm), >99%(280 nm). Conditions: Zorbax C-18, gradient 20-50% B in 25 min, flow: 1mL/min, 25° C., A: 0.1% TFA in H₂O, B:0.1% TFA in CH₃CN; ChiralPurity: >99% (215 nm)>99% (254 nm)>99% (280 nm), Retention time: 20.31min; Chiral HPLC Conditions: Chiralpak AD, 30% ethanol/70% hexane.Rotation: [α]_(D)=+9.13° (c=1.06, MeOH).

Compound 20 Methyl3-{(R)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-4-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

To a solution of optically pure INTERMEDIATE 4a (0.150 g, 0.35 mmol) in4 ml of DMF was added K₂CO₃ (0.122 g, 0.88 mmol) and4-(chloromethyl)-1,3-thiazole (0.066 g, 0.39 mmol) and the mixture washeated at 60° C. overnight. The solvent was evaporated and the crudematerial was dissolved in dichloromethane, washed with water then brine.The organic extract was dried over Na₂SO₄, filtered and concentrated togive crude product that was purified by reverse phase HPLC (gradient5-50% CH₃CN in H₂O containing 0.1% TFA) to give COMPOUND 20 (0.030 g,11.3% yield) as the TFA salt. This material was lyophilized fromCH₃CN/H₂O to produce white powder. ¹H NMR (400 MHz, CD₃OD) δ 1.09 (t,J=6.6 Hz, 3H), 1.22 (t, J=6.8 Hz, 3H), 2.36 (br s., 2H), 3.04 (br s.,2H), 3.19-3.35 (m, 4H), 3.42 (br s., 2H), 3.51 (q, J=6.6 Hz, 2H), 3.72(s, 3H), 4.45 (s, 1H), 4.52 (s, 2H), 7.10 (dt, J=1.5, 7.0 Hz, 1H),7.16-7.24 (m, 2H), 7.33 (d, J=8.4 Hz, 2H), 7.56 (d, J=8.2 Hz, 2H), 7.71,(s, 1H), 7.84 (s, 1H), 9.1 (s, 1H). Purity (HPLC): >89% (215 nm), >99%(254 nm), >99% (280 nm); Conditions: Zorbax C-18, gradient: 10-95% B in25 min, flow: 1 mL/min, 40° C., A—0.1% Formic Acid in H₂O, B—0.1% FormicAcid in MeCN; Chiral purity: >99% (215 nm), >99% (254 nm), >99% (280nm), Retention time 6.67 min; Conditions: Chiralpak AD 50% IPA/50%hexane. Rotation [α]¹⁸ _(D)=−14.7° (c=0.88, MeOH)

Compound 21 Methyl3-{(S)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-4-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

To a solution of optically pure INTERMEDIATE 4b (0.225 g, 0.53 mmol) in4 ml of DMF was added K₂CO₃ (0.183 g, 1.33 mmol) and4-(chloromethyl)-1,3-thiazole (0.099 g, 0.58 mmol) and the mixture washeated at 60° C. overnight. The solvent was evaporated and the crudematerial was dissolved in dichloromethane, washed with water then brine.The organic extract was dried over Na₂SO₄, filtered and concentrated togive crude product that was purified by reverse phase HPLC (gradient5-50% CH₃CN in H₂O containing 0.1% TFA) to give COMPOUND 21 (0.166 g,41.7% yield) as the TFA salt. This material was lyophilized fromCH₃CN/H₂O to produce white powder. ¹H NMR (400 MHz, CD₃OD) δ 0.97 (t,J=7.2 Hz, 3H), 1.11 (t, J=6.8 Hz, 3H), 2.25 (br s., 2H), 2.91 (br s.,2H), 3.07-3.21 (m, 4H), 3.31 (br s., 2H), 3.41 (q, J=6.8 Hz, 2H), 3.61(s, 3H), 4.34 (s, 1H), 4.41 (s, 2H), 6.99 (dt, J=1.6, 7.0 Hz, 1H),7.05-7.13 (m, 2H), 7.22 (d, J=8.2 Hz, 2H), 7.45 (d, J=8.2 Hz, 2H), 7.60(s, 1H), 7.74 (d, J=2.0 Hz, 1H), 8.99 (d, J=2.0 Hz, 1H), 9.13 (s, 1H).

Anal. calcd for C₂₈H₃₅N₅O₃S×1.8TFA×1.1H₂O: C, 50.83; H, 5.26; N, 9.38.Found: C, 50.82; H, 5.23; N, 9.33. MS (calcd): 552.2 (MH+), MS (found):552.2 (MH+). Purity (HPLC): >99% (215 nm), >99% (254 nm), >99% (280 nm);Conditions: Zorbax C-18, gradient: 10-95% B in 25 min, flow: 1 mL/min,40° C., A—0.1% Formic Acid in H₂O, B—0.1% Formic Acid in MeCN; Chiralpurity: >99% (215 nm), >99% (254 nm), >99% (280 nm), Retention time 6.66min; Conditions: Chiralpak AD 50% IPA/50% hexane. Rotation [α]¹⁸_(D)=+14.5° (c=1.07, MeOH)

Compound 22 Methyl3-{(R)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-5-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

To a solution of INTERMEDIATE 4a (0.163 g, 0.38 mmol) in 1,2dichloroethane (6 ml) was added thiazole-5-carboxaldehyde (0.087 g, 0.77mmol) and NaHB(OAc)₃ (0.163 g, 0.77 mmol). The reaction was stirredovernight at room temperature and was quenched with saturated aqueousNaHCO₃. The layers were separated and the aqueous layer was extractedwith dichloromethane (2×15 mL). The combined organic layers were washedwith H₂O, brine, then dried over Na₂SO₄, filtered, concentrated andpurified by reverse phase HPLC (gradient 5-50% CH₃CN in H₂O containing0.1% TFA) to give COMPOUND 22 (0.205 g, 71.2% yield) as the TFA salt.This material was lyophilized from CH₃CN/H₂O to produce a pale yellowsolid. ¹H NMR (400 MHz, CD₃OD) δ 1.09 (t, J=6.8 Hz, 3H), 1.22 (t, J=6.8Hz, 3H), 2.77 (br s., 4H), 3.18-3.40 (m, 6H), 3.51 (q, J=7.0 Hz, 2H),3.72 (s, 3H), 4.56 (s, 1H), 4.63 (s, 2H), 7.12 (dt, J=1.9, 8.4 Hz, 1H),7.19 (dt, J=1.95, 8.4 Hz, 1H), 7.21-7.26 (m, 1H), 7.34 (d, J=8.4 Hz,2H), 7.57 (d, J=8.2 Hz, 2H), 7.74 (s, 1H), 8.05 (s, 1H), 9.17 (s, 1H).Anal. calcd for C₂₈H₃₅N₅O₃S×2.6TFA×0.3H₂O: C, 48.42; H, 4.68; N, 8.50.

Found: C, 48.49; H, 4.83; N, 8.30. MS (calcd): 552.2 (MH+), MS (found):552.2 (MH+). Purity (HPLC): >99% (215 nm), >99% (254 nm), >99% (280 nm);Conditions: Zorbax C-18, gradient: 10-95% B in 25 min, flow: 1 mL/min,40° C., A—0.1% Formic Acid in H₂O, B—0.1% Formic Acid in MeCN; Chiralpurity: >99% (215 nm), >99% (254 nm), >99% (280 nm), Retention time 9.92min; Conditions: Chiralpak AD 50% IPA/50% hexane. Rotation [α]¹⁸_(D)=−12.4° (c=1.01, MeOH).

Compound 23 Methyl3-{(S)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-5-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate

To a solution of INTERMEDIATE 4b (0.223 g, 0.53 mmol) in 1,2dichloroethane (8 ml) was added thiazole-5-carboxaldehyde (0.119 g, 1.05mmol) and NaHB(OAc)₃ (0.223, 1.05 mmol). The reaction was stirredovernight and quenched with saturated aqueous NaHCO₃. The layers wereseparated and the aqueous phase was extracted with dichloromethane (2×15mL). The combined organic extracts were washed with H₂O, brine, driedover Na₂SO₄, filtered, concentrated and purified by reverse phase HPLC(gradient 5-50% CH₃CN in H₂O containing 0.1% TFA) to give COMPOUND 23(0.214 g, 54.2% yield) as the TFA salt. This material was lyophilizedfrom CH₃CN/H₂O to produce a yellow solid. ¹H NMR (400 MHz, CD₃OD) δ 1.09(t, J=6.3 Hz, 3H), 1.22 (t, J=6.6 Hz, 3H), 2.52 (br s., 2H), 2.97 (brs., 2H), 3.18-3.40 (m, 6H), 3.51 (q, J=6.8 Hz, 2H), 3.72 (s, 3H), 4.56(s, 1H), 4.64 (s, 2H), 7.11 (dt, J=1.56, 7.4 Hz, 1H), 7.17-7.21 (m, 1H),7.21-7.26 (m, 1H), 7.35 (d, J=8.2 Hz, 2H), 7.57 (d, J=8.4 Hz), 7.74 (s,1H), 8.05 (s, 1H), 9.17 (s, 1H). Purity (HPLC): >98% (215 nm), >99% (254nm), >99% (280 nm); Conditions: Zorbax C-18, gradient: 10-95% B in 25min, flow: 1 mL/min, 40° C., A—0.1% Formic Acid in H2O, B—0.1% FormicAcid in MeCN; Chiral purity: >99% (215 nm), >99% (254 nm), >99% (280nm), Retention time 13.26 min; Conditions: Chiralpak AD 50% IPA/50%hexane. Rotation [α]¹⁸ _(D)=+12.7° (c=0.97, MeOH).

1. A method for the therapy of anxiety in a warm-blooded animal,comprising: administering to said animal in need of such therapy atherapeutically effective amount of a compound according to formula I,or a pharmaceutically acceptable salt, diastereomer, or enantiomerthereof, or mixtures thereof:

wherein R¹ is —H or —CH₂—R⁴; R² is —H or C₁₋₃alkyl; R³ is C₁₋₆alkyl orC₃₋₆cycloalkyl; and R⁴ phenyl; pyridyl; thienyl; furyl; imidazolyl;triazolyl; pyrrolyl; thiazolyl; or N-oxido-pyridyl, wherein said phenyl;pyridyl; thienyl; furyl; imidazolyl; triazolyl; pyrrolyl; thiazolyl; andN-oxido-pyridyl are optionally substituted with one or more groupsselected from C₁₋₆alkyl, halogenated C₁₋₆alkyl, —NO₂, —CF₃, C₁₋₆ alkoxy,chloro, fluoro, bromo, and iodo.
 2. A method according to claim 1,wherein R¹ is —CH₂—R⁴; R² is —H or C₁₋₃alkyl; R³ is C₁₋₆alkyl orC₃₋₆cycloalkyl; and R⁴ phenyl; pyridyl; thienyl; furyl; imidazolyl;triazolyl; pyrrolyl; thiazolyl; or N-oxido-pyridyl, wherein said phenyl;pyridyl; thienyl; furyl; imidazolyl; triazolyl; pyrrolyl; thiazolyl; andN-oxido-pyridyl are optionally substituted with one or more groupsselected from C₁₋₆alkyl halogenated C₁₋₆alkyl —NO₂, —CF₃, C₁₋₆ alkoxychloro, fluoro, bromo, and iodo.
 3. A method according to claim 2,wherein R⁴ is phenyl; pyridyl; thienyl; furyl; imidazolyl; pyrroly; orthiazolyl; R² is —H or methyl; and R³ is methyl; ethyl; propyl; orisopropyl.
 4. A method according to claim 1, wherein R¹ is —H; R² is —Hor C₁₋₃alkyl; and R³ is C₁₋₆alkyl or C₃₋₆cycloalkyl.
 5. A methodaccording to claim 1, wherein the compound according to formula I isselected from: Methyl3-[(4-[(diethylamino)carbonyl]phenyl)(4-benzyl-piperazin-1-yl)methyl]phenylcarbamate;Methyl-3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(thien-3-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(2-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(3-furylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(1H-imidazol-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(pyridin-2-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]-phenyl}[4-(pyridin-4-yl-methyl)piperazin-1-yl}methyl}phenylcarbamate;Methyl3-{{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-2-ylmethyl)-piperazin-1-yl]methyl}phenylcarbamate;[3-[[4-[(diethylamino)carbonyl]phenyl][4-(phenylmethyl)-1-piperazinyl]methyl]phenyl]-carbamicacid methyl ester;[3-[(S)-[4-[(diethylamino)carbonyl]phenyl][4-(3-pyridinylmethyl)-1-piperazinyl]methyl]phenyl]-carbamicacid, methyl ester;[3-[(S)-[4-[(diethylamino)carbonyl]phenyl][4-(2-thiazolylmethyl)-1-piperazinyl]methyl]phenyl]-carbamicacid, methyl ester; Methyl3-{(R)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-4-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{(S)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-4-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{(R)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-5-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;Methyl3-{(S)-{4-[(diethylamino)carbonyl]phenyl}[4-(1,3-thiazol-5-ylmethyl)piperazin-1-yl]methyl}phenylcarbamate;and[3-[[4-[(diethylamino)carbonyl]phenyl]-1-piperazinylmethyl]phenyl]-carbamicacid, methyl ester; and enantiomers and pharmaceutically acceptablesalts thereof.